Sample records for somatic embryogenesis induced

COMPARED with indirect somaticembryogenesis, direct somaticembryogenesis appears to be associated with greater genetic and cytological uniformity, and it takes less time to induce direct embryogenesis than indirect embryogenesis. Although cotton somaticembryogenesis has been reported by many scientists, direct somaticembryogenesis has not been reported yetr'; . This letter first describes the induction of direct somaticembryogenesis and

The present research investigates the possibility that three diphenylurea (DPU) derivatives, N-phenyl-N?-benzothiazol-6-ylurea (PBU), N,N?-bis-(2,3-methilendioxyphenyl)urea (2,3-MDPU) and N,N?-bis-(3,4-methilendioxyphenyl)urea (3,4-MDPU), stimulate the induction of somaticembryogenesis in three Citrus species. The hypothetical embryogenic activity was assessed using stigma and styles of Citrus myrtifolia Raf., Citrus madurensis Lour. and Citrus limon (L.) Burm. The three compounds influenced the production of somatic embryos differently

NAA and 2,4-D were compared for their ability to inducesomaticembryogenesis in cassava (Manihot esculenta Crantz). In all seven cultivars tested, only 2,4-D had the capacity to induce primary somatic embryos from leaf explants,\\u000a however, both NAA and 2,4-D were capable of inducing secondary somatic embryos. More secondary somatic embryos were formed\\u000a in NAA than in 2,4-D medium. Furthermore,

Summary Somaticembryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly

Common bean Phaseolus vulgaris L. has been shown to be a recalcitrant plant to inducesomaticembryogenesis (SE) under in vitro conditions. We used an alternative strategy to induce SE in common bean based upon the use of a cytokinin (BAP) coupled with osmotic stress adaptation instead of SE response that is induced by auxins. Explants derived from zygotic embryos of common bean were subjected to osmotic stress (sucrose 12 % w/v, 0.5 M) in the presence of BAP 10 mg/L and adenine free base 40 mg/L to inducesomatic embryos from specific competent cells of the apical meristem and cotyledonary node. Somatic embryos were obtained from the competent cells in a direct response (direct SE). In a secondary response (secondary SE), those somatic embryos formed proembryogenic masses (PEM) that originated/developed into secondary somatic embryos and showed the SE ontogeny. Maturation of somatic embryos was achieved by using different osmolality media and converted to plants. Full-visible light spectrum was necessary to achieve efficient plant regeneration. Long-term recurrent SE was demonstrated by propagation of PEM at early stages of SE. This protocol is currently being applied for stable genetic transformation by means of Agrobacterium tumefaciens and bioballistics as well as for basic biochemical and molecular biology experiments. PMID:25252886

This study describes a successful method of somaticembryogenesis and genetic transformation using immature cotyledons of Prunus mume. Immature cotyledons from four different developmental stages of eight different P. mume cultivars were used for the experiments to optimize somaticembryogenesis and genetic transformation protocols. Somaticembryogenesis was induced when the explants were cultured on somatic embryo inducing medium consisting of

We have developed a reproducible system for efficient direct somaticembryogenesis from leaf and internodal explants of Paulownia elongata. The somatic embryos obtained were subsequently encapsulated as single embryos to produce synthetic seeds. Several plant growth regulators [6-benzylaminopurine, indole-3-acetic acid, a-naphthaleneacetic acid, kinetin and thidiazuron (TDZ)] alone or in combination were tested for their capacity to inducesomaticembryogenesis. The

The subject of this study is inducingsomaticembryogenesis in the callus of Lyciumbarbarum L. and determining hydrogen peroxide in somaticembryogenesis. First of all, the activities of three antioxidant enzymes (SOD, peroxidase, catalase) in different stages of somaticembryogenesis were determined. The result showed that the activity of SOD gradually increased in the early days of differentiation culture and

The subject of this study was inducingsomaticembryogenesis in the callus of Lycium barbarum L., The uptake rate of several metal ions in somaticembryogenesis was investigated by multitracer techniques. It was found that some metal ions changed the somaticembryogenesis dynamically. The uptake rates of metal ions were different from each other and exert mutual effect, mutual influence

Energy-dispersive X-ray fluorescence technique (EDXRF) has been extensively used to characterize trace element profiles during plant growth under stress and development. In this study, elemental accumulation was analyzed using EDXRF technique during somaticembryogenesis, from de-differentiated callus (S1) to proembryogenic callus (S2), embryogenic callus with developing embryos (S3) and embryo converted plantlets (S4, S5). There was much variation in Mg, K, Ca, Mn, Fe, Cu and Zn. Higher Mg (4.6%) K (1068 ppm) and Fe accumulation was observed in proembryogenic callus (S2) stage compared to other stages suggesting specific elemental accumulation in embryogenic callus. The results suggest that the information on the accumulation of elements during developmental stages in vitro could be useful for formulating a media for induction of high frequency of embryogenesis in sugarcane.

Somaticembryogenesis and plant regeneration were induced from immature embryonal axes and immature cotyledons of peanut (Arachis hypogaea L. fastigata type cv JLM-1). Influence of different auxins, cytokinins and sugars on somaticembryogenesis from immature cotyledon explants was also investigated. Among the different auxins tested, 2,4-dichlorophenoxyacetic acid (2,4-d) was most effective, producing the highest frequency of responding cultures and highest

AnAgrobacterium rhizogenes-mediated procedure for transformation of papaya (Carica papaya) was developed. Transgenic plants were obtained from somatic embryos that spontaneously formed at the base of transformed\\u000a roots, induced from leaf discs infected withA. rhizogenes. Transformation was monitored by autonomous growth of roots and somatic embryos, resistance to kanamycin, ?-glucuronidase\\u000a activity (GUS), and Southern hybridization analysis. Over one-third of the infected

Direct exposure of calluses of Lycium barbarum L. to an auxin-free medium can inducesomaticembryogenesis. Somatic\\u000a \\u000a embryogenesis of Lycium barbarum L. is controlled artificially by regulating 2,4-D concentration. The total RNA that was isolated from calluses, embryonic\\u000a calluses and early somatic embryos was used for analyzing differential genes expression. We obtained three cDNAs from early\\u000a somaticembryogenesis which were

Somatic embryos were initiated with mature seeds of neem (Azadirachta indica A. Juss.) when cultured on Murashige and Skoog's medium supplemented with thidiazuron (TDZ). Regeneration occurred via somatic\\u000a embryogenesis: direct embryo formation and through an intermediary callus phase. TDZ was very effective and inducedsomatic\\u000a embryogenesis across a wide range of concentrations (150 µm). However, somaticembryogenesis was accompanied by

An efficient and reproducible plant regeneration system, initiated in somatic tissues, has been devised for cassava (Manihot esculenta Crantz). Somaticembryogenesis has been induced from shoot tips and immature leaves of in vitro shoot cultures of 15 cassava genotypes. Somatic embryos developed directly on the explants when cultured on a medium containing 416 mg\\/l 2,4-D. Differences were observed with respect

Somatic embryos isolated from mature seed-derived cotyledon cultures of cassava (Mannihot esculenta Crantz) underwent direct secondary somaticembryogenesis or plant development under appropriate incubation conditions. Isolated somatic embryos were subjected to a two-stage culture procedure similar to that which induced their development on cotyledon explants. This involved incubation for 2430 days on Murashige and Skoog basal medium supplemented with 28

The objective of this study was to inducesomaticembryogenesis in Hedychium bousigonianum Pierre ex Gagnepain and assess the influence of salicylic acid (S) on somaticembryogenesis. Somatic embryos and subsequently regenerated plants were successfully obtained 30 days after transfer of embryogenic...

Indirect somaticembryogenesis was induced on leaf explants of greenhouse-grown Helianthus maximiliani plants. Leaves of the regenerated plants were used as starting explants for the induction of direct somaticembryogenesis.\\u000a Another cycle of somaticembryogenesis was induced on the leaves of regenerated plants. In both cases, leaf explants were\\u000a cultured on media containing different auxin\\/cytokinin ratios. The auxin\\/cytokinin ratio had

Indirect somaticembryogenesis was obtained inPrunus avium L. from either somatic or zygotic embryos. An embryogenic line\\u000a was established by reinduction of embryogenic calluses from somatic embryos. The line was maintained for more than 3 years\\u000a through 6 generations of embryogenic cultures. In the last 2 generations, more than 50% of the explants were embryogenic.\\u000a Embryos at different stages of

This is the first report describing culture conditions necessary to induce secondary embryogenesis in two carnation cultivars,\\u000a Nelson and Spirit. In the first step, embryogenic calli were induced on petal explants followed by development of primary\\u000a somatic embryos from the calli. In the second stage, secondary somatic embryos were obtained when precotyledonary and cotyledonary\\u000a primary embryos were isolated and transferred

Somaticembryogenesis was achieved in four leguminous tree species, that is, Acacia catechu, Acacia arabica, Hardwickia binata, and Dalbergia sissoo using immature zygotic embryos as explants on Murashige and Skoog (MS) medium supplemented with 0.251.0?mg/l Kn (kinetin) and 2.03.0?mg/l 2,4-D (2,4-dichlorophenoxyacetic acid) or NAA (1-napthaleneacetic acid) and 3% sucrose. MS medium containing 2.0?mg/l 2,4-D and 1.01.5?mg/l Kn was noted to be most effective in inducing friable embryogenic callus (FEC); the number of somatic embryos per culture varied in MS medium supplemented with 1.02.0?mg/l 2,4-D or NAA and 0.251.5?mg/l kinetin. The maximum number of somatic embryos was obtained in MS medium containing 1.52.0?mg/l 2,4-D or NAA and 1.01.5?mg/l kinetin; proliferation of embryogenic calli was enhanced in cultures having 1.02.0?mg/l 2,4-D, 1.01.5?mg/l kinetin, and 400600?mg/l L-Proline. The somatic embryos in various shapes and sizes after the first subculture on MS medium supplemented with 0.1?mg/l IAA and 0.25?mg/l BA; developed shoots and rooted in 1/2 strength MS medium supplemented with 0.1?mg/l IBA or IAA. The somatic embryo-derived plantlets were transferred to the field after being hardened in the climate-controlled hardening chamber. PMID:21350667

Somatic embryos were obtained from 1% of cotyledon pieces and hypocotyls of mature embryos of Eucalyptus globulus Labill. cultured on media containing a high concentration of picloram or IBA. 2,4-D and other synthetic auxins did not yield somatic embryos or embryogenic callus. Somatic embryos arose indirectly via callus, being visible after four months, and directly, where little callus or adventitious

Somaticembryogenesis was induced from phylloclade explants of Schlumbergera truncata cv. Russian Dancer. Callus developed on phylloclade explants and sub-cultured over a period of 16 months on MS medium containing\\u000a mainly cytokinins was superior for the induction of somatic embryos compared to callus grown for a shorter time in the establishment\\u000a medium. Sub-culture of callus grown in SH-or MS-based liquid media

SUMMARY The traditional sexual and asexual methods of cocoa propagation have been widely used in Venezuela and worldwide, however, they have not been successful since plants with undesirable agronomic characteristics are produced. At present, plant regeneration via somaticembryogenesis provides an alternative method for clonally propagating cocoa. Therefore, a protocol was developed in order to use staminode, petal, anther and

An efficient primary somatic embryo (SE) and secondary somatic embryo (SSE) production system was developed for the ornamental ginger Hedychium bousigonianum Pierre ex Gagnepain. Addition of two ethylene inhibitors, salicylic acid (SA) and silver nitrate (AgNO3), to the culture media improved the sy...

Summary A cell suspension of French Sombre plantain banana (Musa spp. AAB genome) was initiated from callus obtained from young male flowers. Histological examination enabled us to describe and\\u000a follow the evolution of the suspension consisting of: embryogenic aggregates, proembryos, nodules, and isolated cells. It\\u000a demonstrated the unicellular origin of somatic embryos, either during maintenance of the suspension or after plating

A simple method to inducesomaticembryogenesis from seeds of rapid-cycling Brassica napus is described. Seedlings cultured on Murashige and Skoog (MS) basal medium produced somatic embryos directly on hypocotyls\\u000a and cotyledons after 2 to 3 subcultures onto the same medium. A low pH of the medium (3.55) was more conducive to somatic\\u000a embryogenesis than a higher pH (6 and

A highly efficient system for direct somaticembryogenesis is described. Leaf sections originating from young trifoliate leaves of Medicago falcata line 47\\/15 and Medicago sativa line No2\\/9R, directly produced embryos after cultivation in liquid B5IV induction medium. In comparison with indirect somaticembryogenesis the system omits the callus stage and thus allows shortening of the process of somaticembryogenesis in

The purpose of this study was to improve the somatic embryo (SE) system for plant production of sweetpotato Ipomoea batatas L.(Lam)l. Explants isolated from SE-derived sweet potato plants were compared with control (non SE-derived) plants for their competency for SE production. Leaf explants were cultured on Murashige-Skoog (MS) medium with 2,4-dichlorophenoxy acetic acid (0.2 mg/L) and 6-benzylaminopurine (2.5 mg/L) for 2 weeks in darkness and transferred to MS medium with abscisic acid (2.5 Explants isolated from those plants developed through somaticembryo-genesis produced new somatic embryos rapidly and in higher frequency than those isolated from control plants. They also appeared to grow faster in tissue culture than the control plants. Current studies in the laboratory are examining whether plants derived from a cyclical embryogenesis system (five cycles) would have any further positive impact on the rapidity and frequency of somatic embryo development. More detailed studies using electron microscopy are expected to show the point of origin of the embryos and to allow determination of their quality throughout the cyclical process. This study may facilitate improved plant micropropagation, gene transfer and germplasm conservation in sweet potato.

Summary This report describes a protocol for regeneration ofBrassica nigra in vitro from unorganized callus to a highly differentiated stage of flowering. Callus is initiated from seedling hypocotyl,\\u000a and root explants and plantlets are obtained via somaticembryogenesis. Shoot cultures can be established from these plantlets.\\u000a These shoots can either be induced to flower in vitro or rooted to produce plants

In-vitro induction of somaticembryogenesis and regeneration ability of jojoba (Simmondsia chinensis (Link) Schneider) plant were investigated using two different types of explants, i.e., immature zygotic embryos and leaf disks, and different culture media. Compact embryogenic callus was induced on Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and sucrose. Embryogenic callus was developed from

Proton induced X-ray emission was used to study the trace elemental profiles of embryogenic and non-embryogenic callus of an important cash crop of India - Plantago ovata. Somaticembryogenesis, a well-known process for plant regeneration and crop improvement is modulated by various factors such as ionizing radiation and micro nutrients in the growth media. The present work reports the trace element variation in normal and irradiated callus tissue of P. ovata. Embryogenic and non-embryogenic callus tissues were exposed to gamma rays from a 60Co gamma source. The absorbed dose ranged from 10 to 100 Gy. Subsequent experiments showed significant dose dependent alterations in K, Ca, Mn, Fe, Ni, Cu, Zn, Br, Sr in both the embryogenic and non-embryogenic callus. The precise involvement of these elements has been discussed in light of somaticembryogenesis of the selected medicinal plant.

Programmed cell death (PCD) is a universal process in all multicellular organisms. It is a critical component in a diverse number of processes ranging from growth and differentiation to response to stress. Somaticembryogenesis is one such process where PCD is significantly involved. Nitric oxide is increasingly being recognized as playing a significant role in regulating PCD in both mammalian and plant systems. Plant hemoglobins scavenge NO, and evidence is accumulating that events that modify NO levels in plants also affect hemoglobin expression. Here, we review the process of PCD, describing the involvement of NO and plant hemoglobins in the process. NO is an effector of cell death in both plants and vertebrates, triggering the cascade of events leading to targeted cell death that is a part of an organism's response to stress or to tissue differentiation and development. Expression of specific hemoglobins can alter this response in plants by scavenging the NO, thus, interrupting the death process. Somaticembryogenesis is used as a model system to demonstrate how cell-specific expression of different classes of hemoglobins can alter the embryogenic process, affecting hormone synthesis, cell metabolite levels and genes associated with PCD and embryogenic competence. We propose that plant hemoglobins influence somaticembryogenesis and PCD through cell-specific expression of a distinct plant hemoglobin. It is based on the premise that both embryogenic competence and PCD are strongly influenced by cellular NO levels. Increases in cellular NO levels result in elevated Zn(2+) and reactive-oxygen species associated with PCD, but they also result in decreased expression of MYC2, a transcription factor that is a negative effector of indoleacetic acid synthesis, a hormone that positively influences embryogenic competence. Cell-specific hemoglobin expression reduces NO levels as a result of NO scavenging, resulting in cell survival. PMID:23987809

An alternative in vitro protocol for embryo induction directly from intact living seedlings of Phalaenopsis aphrodite subspecies formosana was established in this study. Without the supplementation of plant growth regulators (PGRs), no embryos were obtained from all the seedlings when cultured on the solid medium. In contrast, embryos formed from the seedlings on the 2-layer medium and the 2-step culture system without the use of PGRs. It was found that the age of the seedlings affected embryo induction. The 2-month-old seedlings typically had higher embryogenic responses when compared with the 4-month-old seedlings in the 2-layer medium or 2-step system. For the 2-month-old seedlings, 1?mg/L TDZ resulted in the highest number of embryos at the distal site of the shoot. However, on the leaves' surface, 0.5?mg/L TDZ induced the highest number of embryos. When the 2-month-old seedlings were cultured using the 2-step method at 1?mg/L of TDZ, the highest embryogenic response was obtained, with an average of 44 embryos formed on each seedling. These adventitious embryos were able to convert into plantlets in a PGR-free 1/2 MS medium, and the plantlets had normal morphology and growth. PMID:24963505

Direct exposure of calluses of Lycium barbarum L. to an auxin-free medium can inducesomaticembryogenesis. The effect of hydrogen peroxide on synthesis of proteins during somaticembryogenesis in Lycium barbarum L. was studied. One-dimensional gel electrophoresis showed that new protein was synthesized by embryogenic callus II (in MS+200 µmol l-1 H2O2 medium). Therefore, we suggested that there was a

Direct exposure of calluses of Lycium barbarum L. to an auxin-free medium can inducesomaticembryogenesis. The effect of hydrogen peroxide on synthesis of proteins during somaticembryogenesis in Lycium barbarum L. was studied. One-dimensional gel electrophoresis showed that new protein was synthesized by embryogenic callus II (in MS+200 µmol l?1 H2O2 medium). Therefore, we suggested that there was a

Secondary somaticembryogenesis and shoot organogenesis from primary somatic embryos of Papaver somniferum L. are described.\\u000a The embryos became malformed, the root meristem expressed dividing activity without position-dependent cell differentiation,\\u000a causing abnormal development or arrested growth of primary somatic embryos. The adventitious shoots regenerated from embryo\\u000a hypocotyl, but secondary somatic embryos had an epidermal origin close to the root meristem.

GENETIC VARIATION IN SOMATICEMBRYOGENESIS OF ROSA HYBRIDA L. A Thesis by ANNA MILDRED BURRELL Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements... for the degree of MASTER OF SCIENCE December 2003 Major Subject: Horticulture GENETIC VARIATION IN SOMATICEMBRYOGENESIS OF ROSA HYBRIDA L. A Thesis by ANNA MILDRED BURRELL Submitted to Texas A...

Somaticembryogenesis is the process by which somatic cells, under induction conditions, generate embryogenic cells, which go through a series of morphological and biochemical changes that result in the formation of a somatic embryo. Somaticembryogenesis differs from zygotic embryogenesis in that it is observable, its various culture conditions can be controlled, and a lack of material is not a

Investigations were undertaken on the use of somaticembryogenesis to generate cocoa swollen shoot virus (CSSV) disease free clonal propagules from infected trees. Polymerase chain reaction (PCR) capillary electrophoresis revealed the presence of CSSV in all the callus tissues induced from the CSSV-infected Amelonado cocoa trees (T1, T2 and T4). The virus was transmitted to primary somatic embryos induced from

Summary The Carabao or Manila Super mango (Mangifera indica L.), a virtually neglected fruit before the advent of KNO3 flower induction in the early 1970s, is now the third leading Philippine export fruit after banana and pineapple. To apply\\u000a biotechnology for improvement, a reliable embryogenesis and regeneration protocol is required. We have developed a protocol\\u000a for somaticembryogenesis and plantlet regeneration

An efficient somaticembryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somaticembryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1?l). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1?1) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N6-benzyladenine (BAP, 0.75 mg 1?l) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1?l) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somaticembryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further. PMID:23961149

Investigations were undertaken on the use of somaticembryogenesis to generate cocoa swollen shoot virus (CSSV) disease free clonal propagules from infected trees. Polymerase chain reaction (PCR) capillary electrophoresis revealed the presence of CSSV in all the callus tissues induced from the CSSV-infected Amelonado cocoa trees (T1, T2 and T4). The virus was transmitted to primary somatic embryos induced from the infected callus tissues at the rate of 10 (19%), 18 (14%) and 16 (15%) for T1, T2 and T4, respectively. Virus free primary somatic embryos from the infected callus tissues converted into plantlets tested CSSV negative by PCR/capillary electrophoresis 2 years after weaning. Secondary somatic embryos induced from the CSSV-infected primary somatic embryos revealed the presence of viral fragments at the rate of 4 (4%) and 9 (9%) for T2 and T4, respectively. Real-time PCR revealed 23 of the 24 secondary somatic embryos contained no detectable virus. Based on these findings, it is proposed that progressive elimination of the CSSV in infected cocoa trees occurred from primary embryogenesis to secondary embryogenesis. PMID:18294704

Twelve cacao (Theobroma cacao) clones propagated by grafting and somaticembryogenesis and grown on an Ultisol soil were evaluated for five years under intensive management at Corozal, Puerto Rico. Preliminary data showed no significant differences between propagation methods for yield of dry beans ...

Manchurian ash (Fraxinus mandshurica Rupr.) is a valuable hardwood species in Northeast China. In cultures of F. mandshurica, somatic embryos were produced mainly on browned explants. Therefore, we studied the mechanism of explant browning and its relationship with somaticembryogenesis (SE). We used explants derived from F. mandshurica immature zygotic embryo cotyledons as materials. Proteins were extracted from browned embryogenic explants, browned non-embryogenic explants, and non-brown explants, and then separated by 2-dimensional electrophoresis. Differentially and specifically expressed proteins were analyzed by mass spectrometry to identify proteins involved in the browning of explants and SE. Some stress response and defense proteins such as chitinases, peroxidases, aspartic proteinases, and an osmotin-like protein played important roles during SE of F. mandshurica. Our results indicated that explant browning might not be caused by the accumulation and oxidation of polyphenols only, but also by some stress-related processes, which were involved in programmed cell death (PCD), and then induced SE. PMID:26084048

Manchurian ash (Fraxinus mandshurica Rupr.) is a valuable hardwood species in Northeast China. In cultures of F. mandshurica, somatic embryos were produced mainly on browned explants. Therefore, we studied the mechanism of explant browning and its relationship with somaticembryogenesis (SE). We used explants derived from F. mandshurica immature zygotic embryo cotyledons as materials. Proteins were extracted from browned embryogenic explants, browned non-embryogenic explants, and non-brown explants, and then separated by 2-dimensional electrophoresis. Differentially and specifically expressed proteins were analyzed by mass spectrometry to identify proteins involved in the browning of explants and SE. Some stress response and defense proteins such as chitinases, peroxidases, aspartic proteinases, and an osmotin-like protein played important roles during SE of F. mandshurica. Our results indicated that explant browning might not be caused by the accumulation and oxidation of polyphenols only, but also by some stress-related processes, which were involved in programmed cell death (PCD), and then induced SE. PMID:26084048

A highly efficient system for direct somaticembryogenesis is described. Leaf sections originating from young trifoliate leaves of Medicago falcata line 47/1-5 and Medicago sativa line No2/9R, directly produced embryos after cultivation in liquid B5IV induction medium. In comparison with indirect somaticembryogenesis the system omits the callus stage and thus allows shortening of the process of somaticembryogenesis in alfalfa by 35-40 days. It permits the avoidance of secondary changes occurring during the process of dedifferentiation. A modified B5/3H medium containing Polyethylene Glycol 6000 promoted embryo development from globular up to torpedo stage. It was clearly shown that 2.5% Polyethylene Glycol stimulated this process for both H. falcata 47/1-5 and M. sativa No 2/9R. Maturation of torpedo stage embryos was carried out on solidified or liquid abscisic acidcontaining medium. A 30?M abscisic acid concentration was optimal in allowing one embryo to yield one plant. Somatic embryo conversion to plants and plant regeneration was performed on Murashige and Skoog medium. Regenerated plants showed a normal morphology. PMID:24221669

Plant regeneration from totipotent cultured cells or protoplasts is a prerequisite for the often proposed genetic modification of plants through somatic cell genetics, and has been achieved in many species. The cereals (and the rest of the Gramineae) have, however, proved to be extremely unresponsive to in vitro culture techniques1. The most convenient source of plant protoplasts is the leaf

Embryogenic callus formation in different larch species from Siberia (Larix sibirica, L. gmelinii, and L. sukaczewii) was carried out on MSGm medium supplemented with growth regulators (2.4-D and BAP) and followed one and the same scheme: elongation of somatic cells and their asymmetric division with formation of initial and tube cells. The cells of embryo initial underwent sequential divisions and formed embryonic globules which caused the formation of somatic embryos. Somatic embryos became mature and germinated by addition of ABA and PEG into the medium. Long-term proliferating cell lines and regenerant plants were obtained in Sukachev larch and its hybrid with Siberian larch. The success of somaticembryogenesis depended on the genotype of the donor tree. PMID:23401960

Somaticembryogenesis fromTheobroma cacao L. flower buds, as previously reported on five Forastero hybrid genotypes, was tested on several other genotypes, belonging to the three cocoa-tree groups: Forastero, Trinitario and Criollo. The results gave evidence of genotypic efficiencies. Explants were cultivated under two successive conditions: callogenesis and expression media. The morphological and histological responses were different for embryogenic or non-embryogenic

The inter-relationship between exogenous calcium (Ca2+) during cold pretreatment and cold-enhanced somaticembryogenesis was investigated using cell suspension cultures of Astragalus adsurgens Pall. Cell suspension was obtained from embryogenic callus and could be induced to form somatic embryos in the differentiation medium. Suspension cells, after cold-treatment at 8 °C for 2 to 3 wk, displayed an enhanced capacity for somatic

A protocol has been developed for somaticembryogenesis and subsequent plant regeneration in Allium schoenoprasum L. Calli were induced from root sections isolated from axenic seedlings and cultivated on media containing either Murashige\\u000a and Skoogs (MS) or Dunstan and Shorts mineral solution supplemented with 5 ?M 2,4-dichlorophenoxyacetic acid (2,4-D) in\\u000a combination with 6-benzylaminopurine (BA), 6-furfurylaminopurine (Kin) or thidiazuron (TDZ) at 1,

Transfer from complete to 2,4-D free Gamborg's B5-medium efficiently inducedsomaticembryogenesis in Papaver tissue cultures (P. somniferum and P. orientale). Embryogenesis was preceded by a strong temporary accumulation of triacylglycerols. In both tissue cultures large amounts of sanguinarine type alkaloids were present, which disappeared during regeneration in the P. orientale cultures but persisted in the P. somniferum cultures. In

Methods for improving the efficiency of plant recovery from somatic embryos of cassava (Manihot esculenta Crantz) were investigated by optimizing the maturation regime and incorporating a desiccation stage prior to inducing germination. Somatic embryos were induced from young leaf lobes of in vitro grown shoots of cassava on Murashige and Skoog medium with 2,4-dichlorophenoxy acetic acid. After 15 to 20

Induction of high frequency somaticembryogenesis from in vitro culture of isolated scutella has been referred to as the Enhanced Regeneration System (ERS). In the present study, the importance of such factors as genotype, culture medium, explant size and cold pretreatment in the induction of somaticembryogenesis and regeneration response of isolated scutella was assessed in a complete block design

Cocoa breeders and growers continue to face the problem of high heterogeneity between individuals derived from one progeny. Vegetative propagation by somaticembryogenesis could be a way to increase genetic gains in the field. Somaticembryogenesis in cocoa is difficult and this species is considered as recalcitrant. This study was conducted to investigate the phenolic composition of cocoa flowers (the

IN VITRO plant regeneration of Gentiana macrophylla Pall. and determination of gentiopicroside content during somaticembryogenesis are described in the present work. The highest percentage of embryogenic callus formation was observed in Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg/L 6-benzylaminopurine (BA). Calli were subcultured on MS medium containing 1.0 mg/L 2,4-D, 1.0 mg/L BA and 500 mg/L lactalbumin hydrolysate (LH) at intervals of 25 days. A higher frequency of somatic embryo maturation was achieved on MS medium containing B5 vitamins (MB) supplemented with different concentrations of 1-naphthaleneacetic acid (NAA) and BA than with a combination of NAA and kinetin (KT). Addition of AgNO(3) improved maturation of somatic embryos while thidiazuron (TDZ) promoted vitrification. The gentiopicroside contents of embryogenic calli and globular-, heart-, torpedo-, and cotyledon-shaped embryoids were determined by high-performance liquid chromatography (HPLC). Gentiopicroside was not detectable in embryogenic calli, but in all types of somatic embryos. The highest gentiopicroside content was observed in cotyledon-shaped embryoids, reaching more than 12 mg/g dry weight. PMID:19548190

The effect of explant age, plant growth regulators and culture conditions on somaticembryogenesis and rosmarinic acid production\\u000a from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced\\u000a on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS)

Somaticembryogenesis (SE) is the developmental reprogramming of somatic cells toward the embryogenesis pathway and is a notable\\u000a illustration of cell totipotency. To identify genes involved in SE, subtractive polymerase chain reaction (PCR) was performed\\u000a to generate transcripts highly enriched for SE-related genes, using cDNA prepared from a mixture of embryogenic callus and\\u000a preglobular somatic embryos, as the tester, and

In plants, complete embryos can develop not only from the zygote, but also from somatic cells in tissue culture. How somatic cells undergo the change in fate to become embryogenic is largely unknown. Proteins, secreted into the culture medium such as endochitinases and arabinogalactan proteins (AGPs) are required for somaticembryogenesis. Here we show that carrot (Daucus carota) AGPs can

Banana is an important tropical fruit worldwide. Parthenocarpy and female sterility made it impossible to improve banana varieties through common hybridization. Genetic transformation for banana improvement is imperative. But the low rate that banana embryogenic callus was induced made the transformation cannot be performed in many laboratories. Finding ways to promote banana somaticembryogenesis is critical for banana genetic transformation. After tobacco arabinogalactan protein gene NtEPc was transformed into Escherichia coli (DE3), the recombinant protein was purified and filter-sterilized. A series of the sterilized protein was added into tissue culture medium. It was found that the number of banana immature male flowers developing embryogenic calli increased significantly in the presence of NtEPc protein compared with the effect of the control medium. Among the treatments, explants cultured on medium containing 10 mg/l of NtEPc protein had the highest chance to develop embryogenic calli. The percentage of lines that developed embryogenic calli on this medium was about 12.5 %. These demonstrated that NtEPc protein can be used to promote banana embryogenesis. This is the first paper that reported that foreign arabinogalactan protein (AGP) could be used to improve banana somaticembryogenesis. PMID:25227688

White spruce is an important commercial species for reforestation. The success in its propagation through somaticembryogenesis is well documented; however the physiological processes involved are poorly understood and remain unoptimized. The variable quality embryos generated in vitro from the same genotype suggest control at the protein and metabolite level. In order to probe metabolic changes, we have conducted a "metabolic footprinting" study, whereby culture media from growing cells was quantitatively analyzed to determine which metabolites were consumed and excreted. Such experiments are advantageous in that there is no need to quench cellular metabolism or extract intracellular metabolites through time-consuming protocols. In this paper we demonstrate the application of the footprinting assay to somatic embryo cells of white spruce (Picea glauca) using 1D (1)H NMR spectroscopy. We have surveyed embryogenesis metabolism in two types of media, maintenance (MN) and maturation (MT). MN medium does not result in shoot apical meristem (SAM) formation, while MT medium induces the necessary changes leading to fully developed somatic embryos. The two types of media were easily distinguished using metabolomics analysis, namely multivariate pattern recognition statistics (orthogonal partial least squares discriminatory analysis). From this analysis, we have identified numerous compounds involved with branched chain amino acid pathways such as valine and isoleucine. These results are explained on the basis of known metabolic pathways implicated in plant and animal developmental processes, and ultimately implicate altered CoA biosynthesis. PMID:19195904

A direct somaticembryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somaticembryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somaticembryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somaticembryogenesis of a high frequency (87-96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somaticembryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somaticembryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somaticembryogenesis. PMID:23518589

Somaticembryogenesis (SE) has the potential to be the lowest-cost method to rapidly produce large numbers of high-value somatic seedlings with desired characteristics for plantation forestry. At least 24 of the 115-120 known Pinus species can undergo SE. Initiation for most species works best with immature megagametophytes as starting material, although a few pines can initiate SE cultures from isolated mature seed embryos. Successful initiation depends heavily on explant type, embryo developmental stage, and medium salt base. Most first reports of initiation used 2,4-D and BAP or a combination of cytokinins. More recent reports have optimized initiation for many Pinus spp., but still use mostly the combinations of auxin and cytokinins. Initiation can be stimulated with medium supplements including abscisic acid (ABA), brassinosteroids, ethylene inhibitors, gibberellin inhibitors, organic acids, putrescine, specific sugar types (maltose, galactose, D-chiro-inositol, and D-xylose), triacontanol, vitamins (B12, biotin, vitamin E, and folic acid), or manipulation of environmental factors including pH, water potential, cone cold storage, gelling agent concentration, and liquid medium. Embryo development and maturation usually occur best on medium containing ABA along with water potential reduction (with sugars and polyethylene glycol) or water availability reduction (with raised gelling agent increasing gel-strength). Activated carbon and maltose may also improve embryo maturation. The main issues holding SE technology back are related to the high cost of producing a somatic seedling, incurred from low initiation percentages for recalcitrant species, culture loss, and decline after initiation and poor embryo maturation resulting in no or poor germination. Although vast progress has been made in pine SE technology over the past 24 years, fundamental studies on seed and embryo physiology, biochemistry, and gene expression are still needed to help improve the technology to a point where large-scale commercialization is economically viable for a broad range of pine species. PMID:21207275

Having produced the embryos of cactus Copiapoa tenuissima Ritt. forma monstruosa at the globular stage and callus, we investigated the effect of abscisic acid (ABA) in the following concentrations: 0, 0.1, 1, 10, and 100??M on successive stages of direct (DSE) and indirect somaticembryogenesis (ISE). In the indirect somaticembryogenesis process we also investigated a combined effect of ABA (0, 0.1, 1??M) and sucrose (1, 3, 5%). The results showed that a low concentration of ABA (0-1??M) stimulates the elongation of embryos at the globular stage and the number of correct embryos in direct somaticembryogenesis, while a high ABA concentration (10100??M) results in growth inhibition and turgor pressure loss of somatic embryos. The indirect somaticembryogenesis study in this cactus suggests that lower ABA concentrations enhance the increase in calli fresh weight, while a high concentration of 10??M ABA or more changes calli color and decreases its proliferation rate. However, in the case of indirect somaticembryogenesis, ABA had no effect on the number of somatic embryos and their maturation. Nevertheless, we found a positive effect of sucrose concentration for both the number of somatic embryos and the increase in calli fresh weight. PMID:23843737

Protocol for direct somaticembryogenesis from leaf explants of economically important species of Dianthus, viz. D. caryophyllus, D. barbatus and D. chinensis has been developed. Murashige and Skoogs (MS) liquid medium supplemented with 2,4-D (1mg\\/l) was used for direct induction of somaticembryogenesis without an intervening callus phase. Initially globular structures were observed after 21 days of culture of leaf

Pineapple guava (Acca sellowiana) syn. Feijoa sellowiana, a Brazilian indigenous Myrtaceae is under domestication in South Brazil. Previous works showed that this species is responsive to somaticembryogenesis and recalcitrant to conventional methods of clonal propagation. In the present work it was evaluated the role of components of culture medium in the induction and development of somatic embryos. The technology

We describe an efficient procedure to obtain somatic embryos from mature zygotic embryos of Hyoscyamus niger (black henbane). It has several advantages over previous regeneration methods, which are: the use of mature seeds, an average 80% somaticembryogenesis rate and a high (eight-fold higher than the control) plant regeneration frequency. The critical step in this protocol was soaking of the

A study of somaticembryogenesis and rhizogenesis and their influence on production of morphinan alkaloids on two species of opium poppy is presented. We identified the ratios of auxin and cytokinin that caused somaticembryogenesis and rhizogenesis in hypocotyl and cotyledons of Papaver somniferum album and Papaver orientale splendidissimum. The hypocotyls and cotyledons both show somaticembryogenesis in Papaver somniferum album whereas only the cotyledons were embryogenic in Papaver orientale splendidissimum. For rhizogenesis, the most important response is on the cotyledons and leaves in these two species. Histology showed characteristic stages of somatic embryo: Globular, cotyledonous, and heart cotyledonary. High performance liquid chromatography analysis showed that the roots of both species synthesized codeine, thebaine, and papaverine. Morphine was only detected in aerial parts of Papaver somniferum album. Codeine and thebaine were detected in the rhizogenous but no embryonic callus. These results suggest that root organogenesis is causally related to alkaloid biosynthesis. PMID:12488612

Summary Somaticembryogenesis and plant regeneration of banana and plantain cultivars (Musa spp.) were obtained by culturing young male flowers. Multiplication and maintenance of embryogenic cultures were achieved\\u000a by culturing somatic embryos in a temporary immersion system (SIT). A multiplication rate of 40 allowed us to obtain more\\u000a than 6000 somatic embryos after 6 mo. of subculture. Plant recovery frequencies were

Somaticembryogenesis (SE) offers vast potential for the clonal propagation of high-value roses. However, some recalcitrant\\u000a cultivars unresponsive to commonly employed SE-inducing agents and low induction rates currently hinder the commercialization\\u000a of SE technology in rose. Rose SE technology requires improvement before it can be implemented as a production system on a\\u000a commercial scale. In the present work, we assessed

Somaticembryogenesis is a powerful biotechnological tool for the mass production of economically important cultivars. Due to the cellular totipotency of plants, somatic cells under appropriate conditions are able to develop a complete functional embryo. During the induction of somaticembryogenesis, there are different factors involved in the success or failure of the somaticembryogenesis response. Among these factors, the origin of the explant, the culture medium and the in vitro environmental conditions have been the most studied. However, the secretion of molecules into the media has not been fully addressed. We found that the somaticembryogenesis of Coffea canephora, a highly direct embryogenic species, is disrupted by the metabolites secreted from C. arabica, a poorly direct embryogenic species. These metabolites also affect DNA methylation. Our results show that the abundance of two major phenolic compounds, caffeine and chlorogenic acid, are responsible for inhibiting somaticembryogenesis in C. canephora. PMID:26038822

Somaticembryogenesis is a powerful biotechnological tool for the mass production of economically important cultivars. Due to the cellular totipotency of plants, somatic cells under appropriate conditions are able to develop a complete functional embryo. During the induction of somaticembryogenesis, there are different factors involved in the success or failure of the somaticembryogenesis response. Among these factors, the origin of the explant, the culture medium and the in vitro environmental conditions have been the most studied. However, the secretion of molecules into the media has not been fully addressed. We found that the somaticembryogenesis of Coffea canephora, a highly direct embryogenic species, is disrupted by the metabolites secreted from C. arabica, a poorly direct embryogenic species. These metabolites also affect DNA methylation. Our results show that the abundance of two major phenolic compounds, caffeine and chlorogenic acid, are responsible for inhibiting somaticembryogenesis in C. canephora. PMID:26038822

Genetic controls for growth of embryogenic cultures, storage, maturation treatments, germination and cryopreservation in white spruce somaticembryogenesis (SE) were examined. These SE processes were under genetic control but less strongly so than the initiation phase. For all the SE characters examined, variance due to clones within families was significant and often the largest genetic component of variance. This was

Somaticembryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1mM ammonium (NH(4)(+)) as the sole source of nitrogen. Growth of NH(4)(+)-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH(4)(+) medium with 25mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH(4)(+) induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH(4)(+) as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somaticembryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20mM) or Gln (10mM) in combination with NH(4)(+) (1mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin. PMID:21807439

In cell cultures of carrot (Daucus carota L.), somaticembryogenesis can be induced by transferring cells from a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) to one devoid of 2,4-D. Previous analysis of transgenic carrot cells containing the 5' non-coding sequence of the Ri plasmid rolC and a structural gene for bacterial beta-glucuronidase (uidA) has shown that the chimeric gene is actively expressed after induction of somaticembryogenesis. In this study, we demonstrate that activation of the rolC promoter is dependent on the process of embryo development but not on the duration of the cell culture in 2,4-D-free medium. We also analyzed the cis region of the rolC promoter that is responsible for somaticembryogenesis-related activation (SERA), namely relatively low beta-glucuronidase (GUS) activity in calli and proembryogenic masses (PEM) and high GUS activity in heart- and torpedo-stage embryos. When the -255-bp region of the rolC gene was used, SERA was retained. Internal deletions within this -255-bp region did not alter SERA by the rolC promoter. Furthermore, when a rolC promoter fragment (-848 to -94 bp) was fused to the cauliflower mosaic virus (CaMV) 35S core region (-90 to +6 bp), it conferred relatively low GUS activity in calli and PEM but high GUS activity in heart and torpedo embryos. When -848 to -255-bp or -255- to -94-bp fragments of the rolC promoter were fused to the same CaMV 35S core region, GUS activity patterns were not related to somaticembryogenesis. These results suggest that the combination of several regulatory regions in the rolC promoter may be required for SERA in carrot cell cultures. PMID:8016259

Embryogenic tissue culture systems are utilized in propagation and genetic engineering of crop plants, but applications are limited by genotype-dependent culture response. To date, few genes necessary for embryogenic callus formation have been identified or characterized. The goal of this research was to enhance our understanding of gene expression during maize embryogenic tissue culture initiation. In this study, we highlight the expression of candidate genes that have been previously regarded in the literature as having important roles in somaticembryogenesis. We utilized RNA based sequencing (RNA-seq) to characterize the transcriptome of immature embryo explants of the highly embryogenic and regenerable maize genotype A188 at 0, 24, 36, 48, and 72 hours after placement of explants on tissue culture initiation medium. Genes annotated as functioning in stress response, such as glutathione-S-transferases and germin-like proteins, and genes involved with hormone transport, such as PINFORMED, increased in expression over 8-fold in the study. Maize genes with high sequence similarity to genes previously described in the initiation of embryogenic cultures, such as transcription factors BABY BOOM, LEAFY COTYLEDON, and AGAMOUS, and important receptor-like kinases such as SOMATICEMBRYOGENESIS RECEPTOR LIKE KINASES and CLAVATA, were also expressed in this time course study. By combining results from whole genome transcriptome analysis with an in depth review of key genes that play a role in the onset of embryogenesis, we propose a model of coordinated expression of somaticembryogenesis-related genes, providing an improved understanding of genomic factors involved in the early steps of embryogenic culture initiation in maize and other plant species. PMID:25356773

An improved method of direct somaticembryogenesis (SE) was developed in Swertia chirata for the first time using leaves and roots of in vitro-grown young seedlings. In the present study, 2,4-dichlorophenoxyacetic\\u000a acid (2,4-D) was assessed individually and in combination with other auxins, as well as with cytokinin for its effectiveness\\u000a to inducesomatic embryos. Leaf explants with abaxial side in

The effect of exogenously supplied reduced nitrogen and sucrose on high-frequency somaticembryogenesis in petiole-derived tissue cultures of a diploid and a tetraploid regenerable clone of Medicago sativa ssp. falcata was investigated. There was an absolute requirement for ammonium during embryo induction and differentiation, with 5mM being the optimum for induction and 1020 mM the optimum for differentiation of somatic

Indirect somaticembryogenesis was tested as a method for eradication of Grapevine fanleaf virus (GFLV) in three grapevine cultivars. Reverse transcriptase-polymerase chain reaction for GFLV detection was performed on\\u000a tissues sampled at various steps of the embryogenic process: flower explants, embryogenic and non-embryogenic calli, single\\u000a somatic embryos and regenerated plants. The virus was detected in all tested anthers and ovaries,

Comparative analysis of zygotic and somaticembryogenesis of Acca sellowiana showed higher amounts of sucrose, fructose, raffinose, and myo-inositol in zygotic embryos at different developmental stages than in corresponding somatic ones. These differences were\\u000a mostly constant. In general, glucose levels were significantly lower than the other soluble carbohydrates analyzed, showing\\u000a minor variation in each embryo stage. Despite the presence of

In spite of the importance of somaticembryogenesis for basic research in plant embryology as well as for crop improvement and plant propagation, it is still unclear which mechanisms and cell signals are involved in acquiring embryogenic competence by a somatic cell. The aim of this work was to study cellular and molecular changes involved in the induction stage in calli of Agave tequilana Weber cultivar azul in order to gain more information on the initial stages of somaticembryogenesis in this species. Cytochemical and immunocytochemical techniques were used to identify differences between embryogenic and non-embryogenic cells from several genotypes. Presence of granular structures was detected after somaticembryogenesis induction in embryogenic cells; composition of these structures as well as changes in protein and polysaccharide distribution was studied using Coomassie brilliant blue and Periodic Acid-Schiff stains. Distribution of arabinogalactan proteins (AGPs) and pectins was investigated in embryogenic and non-embryogenic cells by immunolabelling using anti-AGP monoclonal antibodies (JIM4, JIM8 and JIM13) as well as an anti-methyl-esterified pectin-antibody (JIM7), in order to evaluate major modifications in cell wall composition in the initial stages of somaticembryogenesis. Our observations pointed out that induction of somaticembryogenesis produced accumulation of proteins and polysaccharides in embryogenic cells. Presence of JIM8, JIM13 and JIM7 epitopes were detected exclusively in embryogenic cells, which supports the idea that specific changes in cell wall are involved in the acquisition of embryogenic competence of A. tequilana. PMID:22270826

Plant hormones play critical roles in the establishment of somaticembryogenesis. During this\\u000a process, somatic plant cells reverse their state of differentiation, acquire pluripotentiality and\\u000a set up a new developmental program. The identification of the regulatory mechanisms that govern\\u000a the key events of somaticembryogenesis requires molecular and genetic investigations. One critical\\u000a issue is how plant hormones and growth regulators act

The effects of glutamine-based dipeptides, glutamine and casein hydrolysate, as well as the deletion of organic nitrogen, were investigated during white spruce [Picea glauca (Moench) Voss] somaticembryogenesis. There were no differences in the fresh weight increase of the tissue masses grown on initiation medium with different combinations of organic nitrogen. This was also the case for subsequent growth on

Cocoa breeders and growers continue to face the problem of high heterogeneity between individuals derived from one progeny. Vegetative propagation by somaticembryogenesis could be a way to increase genetic gains in the field. Somaticembryogenesis in cocoa is difficult and this species is considered as recalcitrant. This study was conducted to investigate the phenolic composition of cocoa flowers (the explants used to achieve somaticembryogenesis) and how it changes during the process, by means of histochemistry and conventional chemical techniques. In flowers, all parts contained polyphenolics but their locations were specific to the organ considered. After placing floral explants in vitro, the polyphenolic content was qualitatively modified and maintained in the calli throughout the culture process. Among the new polyphenolics, the three most abundant were isolated and characterized by 1H? and 13C?NMR. They were hydroxycinnamic acid amides: N?trans?caffeoyl?l?DOPA or clovamide, N?trans?p?coumaroyl?l?tyrosine or deoxiclovamide, and N?trans?caffeoyl?l?tyrosine. The same compounds were found also in fresh, unfermented cocoa beans. The synthesis kinetics for these compounds in calli, under different somaticembryogenesis conditions, revealed a higher concentration under non?embryogenic conditions. Given the antioxidant nature of these compounds, they could reflect the stress status of the tissues. PMID:12933367

Cocoa breeders and growers continue to face the problem of high heterogeneity between individuals derived from one progeny. Vegetative propagation by somaticembryogenesis could be a way to increase genetic gains in the field. Somaticembryogenesis in cocoa is difficult and this species is considered as recalcitrant. This study was conducted to investigate the phenolic composition of cocoa flowers (the explants used to achieve somaticembryogenesis) and how it changes during the process, by means of histochemistry and conventional chemical techniques. In flowers, all parts contained polyphenolics but their locations were specific to the organ considered. After placing floral explants in vitro, the polyphenolic content was qualitatively modified and maintained in the calli throughout the culture process. Among the new polyphenolics, the three most abundant were isolated and characterized by 1H- and 13C-NMR. They were hydroxycinnamic acid amides: N-trans-caffeoyl-l-DOPA or clovamide, N-trans-p-coumaroyl-l-tyrosine or deoxiclovamide, and N-trans-caffeoyl-l-tyrosine. The same compounds were found also in fresh, unfermented cocoa beans. The synthesis kinetics for these compounds in calli, under different somaticembryogenesis conditions, revealed a higher concentration under non-embryogenic conditions. Given the antioxidant nature of these compounds, they could reflect the stress status of the tissues. PMID:12933367

The objectives of this study were to determine the effects of low temperature (4 °C) preincubation on somaticembryogenesis from orchardgrass (Dactylis glomerata L.) leaf cultures and to relate these effects to ethylene emanation during the preincubation and incubation periods. Experiments were also conducted with an ethylene biosynthesis inhibitor aminooxyacetic acid (AOA). Segments from the innermost two leaves were cultured

A procedure for the development of alfalfa (Medicago falcata L.) somatic embryos to the torpedo stage in air-lift vessels is described. Embryos were initiated from chopped leaf explants and were formed by direct somatic embryogensis. The system produced a high number of torpedo stage embryos. The effect of various inoculation densities on embryo development was studied. A procedure for the

Flow cytometry analyses were used to verify the ploidy stability of Quercus suber L. somaticembryogenesis process. Leaf explants of two adult cork oak trees (QsG0 and QsG5) of the North of Portugal were inoculated on MS medium with 2,4-D and zeatin. After 3 months, calluses with embryogenic structures were isolated and transferred to fresh MS medium without growth regulators and somatic embryo evolution was followed. Morphologically normal somatic embryos (with two cotyledons) and abnormal somatic embryos (with one or three cotyledons) were used in this assay. Flow cytometry combined with propidium iodide staining was employed to estimate DNA ploidy levels and nuclear DNA content of somatic embryos and leaves from mother plants. No significant differences (P< or =0.05) were detected among embryos, and between the embryos and the mother plants. Also, after conversion of these embryos, no significant morphological differences were observed among the somatic embryo-derived plants. These results and further studies using converted plantlet leaves and embryogenic callus tissue indicate that embryo cultures and converted plantlets were stable with regard to ploidy level. As no major somaclonal variation was detected our primary goal of "true-to-type" propagation of cork oak using somaticembryogenesis was assured at this level. The estimation of the 2C nuclear DNA content for this species is similar to the previously obtained value. PMID:15744492

Auxin and cytokinin signaling participates in regulating a large spectrum of developmental and physiological processes in plants. The shoots and roots of plants have specific and sometimes even contrary responses to these hormones. Recent studies have clearly shown that establishing the spatiotemporal distribution of auxin and cytokinin response signals is central for the control of shoot apical meristem (SAM) induction in cultured tissues. However, little is known about the role of these hormones in root apical meristem (RAM) initiation. Here, we found that the expression patterns of several regulatory genes critical for RAM formation were correlated with the establishment of the embryonic root meristem during somaticembryogenesis in Arabidopsis. Interestingly, the early expression of the WUS-RELATED HOMEOBOX 5 (WOX5) and WUSCHEL genes was induced and was nearly overlapped within the embryonic callus when somatic embryos (SEs) could not be identified morphologically. Their correct expression was essential for RAM and SAM initiation and embryonic shootroot axis establishment. Furthermore, we analyzed the auxin and cytokinin response during SE initiation. Notably, cytokinin response signals were detected in specific regions that were correlated with induced WOX5 expression and subsequent SE formation. Overexpression of the ARABIDOPSIS RESPONSE REGULATOR genes ARR7 and ARR15 (feedback repressors of cytokinin signaling), disturbed RAM initiation and SE induction. These results provide new information on auxin and cytokinin-regulated apicalbasal polarity formation of shootroot axis during somaticembryogenesis. PMID:25642237

A culture model was developed in Vitis vinifera L., cultivar 'Chardonnay' for studying SE (SomaticEmbryogenesis). The auxin 2,4-D (2,4-Dichlorophenoxyacetic acid) was used to induce indirect secondary embryogenesis at a high rate, starting from embryos derived from embryogenic cultures previously obtained. Cotyledonary embryos were shown to be more responsive to SE induction than embryos at the torpedo-stage and were used for molecular analyses. The expression of SERK (SomaticEmbryogenesis Receptor Kinase), L1L (Leafy Cotyledon1 Like) and a set of PR (Pathogenesis-Related) genes was monitored during the whole SE process. VvSERK1, VvSERK2 and VvL1L were down-regulated by the 2,4-D treatment but expressed in embryonic tissues. On the contrary, VvPR1, VvPR8, VvPR10.1 and VvPR10.3 were strongly up-regulated by the 2,4-D treatment, and their transcripts were not or only weakly detected in clusters of secondary embryos. VvSERK3, VvPR3 and VvPR10.2 were more stably expressed in all tissues examined. The discussion deals with the putative role of the different genes in grapevine SE. PMID:19406655

Molecular mechanisms controlling plant totipotency are largely unknown and studies on somaticembryogenesis (SE), the process through which already differentiated cells reverse their developmental program and become embryogenic, provide a unique means for deciphering molecular mechanisms controlling developmental plasticity of somatic cells. Among various factors essential for embryogenic transition of somatic cells transcription factors (TFs), crucial regulators of genetic programs, are believed to play a central role. Herein, we used quantitative real-time polymerase chain reaction (qRT-PCR) to identify TF genes affected during SE induced by in vitro culture in Arabidopsis thaliana. Expression profiles of 1,880 TFs were evaluated in the highly embryogenic Col-0 accession and the non-embryogenic tanmei/emb2757 mutant. Our study revealed 729 TFs whose expression changes during the 10-days incubation period of SE; 141 TFs displayed distinct differences in expression patterns in embryogenic versus non-embryogenic cultures. The embryo-induction stage of SE occurring during the first 5 days of culture was associated with a robust and dramatic change of the TF transcriptome characterized by the drastic up-regulation of the expression of a great majority (over 80%) of the TFs active during embryogenic culture. In contrast to SE induction, the advanced stage of embryo formation showed attenuation and stabilization of transcript levels of many TFs. In total, 519 of the SE-modulated TFs were functionally annotated and transcripts related with plant development, phytohormones and stress responses were found to be most abundant. The involvement of selected TFs in SE was verified using T-DNA insertion lines and a significantly reduced embryogenic response was found for the majority of them. This study provides comprehensive data focused on the expression of TF genes during SE and suggests directions for further research on functional genomics of SE. PMID:23874927

Establishment, maintenance, regeneration, and transformation of somatic embryos by both direct and indirect means (callus-mediated)\\u000a was achieved for Bixa orellana, a tropical plant whose seeds produce commercially edible annatto pigment, which mainly constitutes an apocarotenoid called\\u000a bixin. Callus-mediated methodology was found to be efficient in producing a greater number of embryos in a short time. The\\u000a maximum of 28 somatic

Two types of flower explants, staminoides and petals, were used for in vitro induction of somatic embryos in cocoa. After 14 days in culture, we observed globular structures and callus formation on both types of explants. However, the better results were obtained on staminoides: 98.3% formed callus and 86.2% somatic embryos on Murashige and Skoog (1962) medium supplemented with sucrose, coconut water, 2,4-D, kinetin and agar. PMID:15952427

Four systems of cyclic somaticembryogenesis of Siberian ginseng (Eleutherococcus senticosus Maxim) were used to study the mechanism of embryonic cell cluster generation. The first, direct somatic embryo induction (DSEI), generates secondary embryos directly from the primary somatic embryos; the second, direct embryogenic cell cluster induction (DEC)), induces embryogenic cell clusters directly from somatic embryos in agar medium. Subsequently, we found that when DEC-derived somatic embryos are transferred to suspension culture or a bioreactor culture, only somatic embryos are induced, and embryogenic cell clusters cannot form. Therefore, these new lines were named DEC cultured by liquid medium (ECS) and DEC cultured by bioreactor (ECB), respectively. Transmission electron microscopy showed that DEC epidermal cells contained a variety of inclusions, distinct from other lines. A cDNA library of DEC was constructed, and 1,948 gene clusters were obtained and used as probes. RNA was prepared from somatic embryos from each of the four lines and hybridized to a microarray. In DEC, 7 genes were specifically upregulated compared with the other three lines, and 4 genes were downregulated. EsXTH1 and EsPLT1, which were among the genes upregulated in DEC, were cloned using the rapid amplification of cDNA ends (RACE). Real-time quantitative PCR showed EsXTH1 was more highly expressed in DEC than in other lines throughout the culture cycle, and EsPLT1 expression in DEC increased as culture duration increased, but remained at a low expression level in other lines. These results suggest that EsXTH1 and EsPLT1 may be the essential genes that play important roles during the induction of embryogenic cell clusters. PMID:24743225

Majority of the Indian soybean cultivars are recalcitrant to tissue culture regeneration. The present communication reports the development of somaticembryogenesis in a liquid culture medium from immature cotyledons of G. max. Following induction with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthalene acetic acid (NAA), the number of somatic embryos and percentage of explants that responded were higher with 45.24 microM 2,4-D. The proliferation of somatic embryos for three successive cycles was achieved in 22.62 microM 2,4-D. Histodifferentiation of somatic embryos under NAA (10.74 microM) indicated that better embryo development and maturation was achieved without any growth regulator. The amino acids such as L-glutamine favoured the somatic embryo induction and histodifferentiation at 20 and 30 mM respectively, where as L-asparagine at 10 mM concentration enhanced the somatic embryo proliferation. In addition, somatic embryos that were desiccated (air-drying method) for 5 days showed better germination (40.88%). The Indian soybean cultivars also showed strict genotypic influence and cv. Pusa 16 was emerged as a best responding cultivar for somatic embryo induction with 74.42% of response. PMID:24266110

An antibiotic, cefotaxime (Omnatax) has been found to promote somaticembryogenesis and subsequent plant regeneration in vitro in indica-type basmati rice cultures. Response was highly genotype specific. The number, mass and morphology of the calli formed on the scutellar tissues were dependent on the growth medium (with or without cefotaxime). The embryogenic nature of nodular calli was confirmed through histological analysis and their plant regeneration ability. The calli of variety Pusa basmati 1 grown on medium supplemented with cefotaxime (100 mg/L) exhibited up to 70.5% plant regeneration as compared to control (51.51%). Plants regenerated from emryogenic calli were phenotypically normal and identical to seed-derived plants and exhibited normal fertility. A limited humidity and an optimal aeration of the culture tubes further enhanced the frequency of somaticembryogenesis and plant regeneration. PMID:17125131

The characterization of cellular changes that occur during somaticembryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora. PMID:21927886

Embryogenic callus was obtained from mature seed explants on medium supplemented with 2,4-dichlorophenoxyacetic acid. Primary somatic embryos (SEs) can only develop into abnormal plants. Well-developed SEs could be obtained through secondary somaticembryogenesis both in solid and liquid cultures. Temperature strongly affected induction frequency of secondary embryogenesis. Relatively high temperature (30°C) and germinated SEs explants were effective for induction of secondary somatic embryos, and low temperature (20°C) was more suitable for further embryo development, plantlet conversion, and transplant survival. Somatic embryos formed on agar medium had larger cotyledons than those of embryos formed in liquid medium. Supplementing 0.1?mg?L(-1) 6-benzyladenine (BA) was effective for plant conversion; the rate of plant conversion was 43.3% in somatic embryos from solid culture and 36.5% in embryos from liquid culture. In vitro plants were successfully acclimatized in the greenhouse. The protocol established in this study will be helpful for large-scale vegetative propagation of this medicinal tree. PMID:23818829

An efficient, highly reproducible system for plant regeneration via somaticembryogenesis was developed for Cenchrus ciliaris genotypes IG-3108 and IG-74. Explants such as seeds, shoot tip segments and immature inflorescences were cultured on Murashige\\u000a and Skoog (MS) medium supplemented with 2.05.0 mg dm?3 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg dm?3 N6-benzyladenine (BA) for induction of callus. Callus could be successfully

Young inflorescence explants of Setaria italica in culture showed high capacity for regenerating plantlets through somaticembryogenesis. Embryogenic callus formation was initiated from the explants cultured on Murashige and Skoog's medium with 2 mg\\/l 2,4-D and 0.20.5 mg\\/l KT or BAP, but it was better for the maintenance of embryogenic growth to subculture the calli on the medium with 2,4-D

Summary An in vitro culture procedure was established for somaticembryogenesis and plant regeneration from callus cultures of the important\\u000a palm betel nut (Areca catechu L.). Segments of zygotic embryos of Areca catechu L. were cultured on Murashige and Skoog basal medium supplemented with dicamba (9.05, 18.1, 27.15, and 36.2 ?M). After 78 wk in darkness, wounded regions of explants formed

Various factors affect the induction of somaticembryogenesis in peach palm (Bactris gasipaes Kunth). Among these, both the type and level of auxins had the greatest influence on in vitro responses, although the genotype\\u000a and the developmental stage of the explants also influenced results. Younger inflorescences were more competent to respond\\u000a to SE induction than more mature inflorescences and the

Nineteen accessions of diploid Medicago sativa L. belonging to the four subspecies sativa, caerula, falcata and xvaria were screened for their ability to produce somatic embryos on hypocotyl-derived callus. Two medium protocols were used in this study, a three-step sequence with exposure of the callus cultures to a high 2,4-D concentration and a two-step sequence without exposure to a high

Nineteen accessions of diploid Medicago sativa L. belonging to the four subspecies sativa, caerula, falcata and xvaria were screened for their ability to produce somatic embryos on hypocotyl-derived callus. Two medium protocols were used in this study, a three-step sequence with exposure of the callus cultures to a high 2,4-D concentration and a two-step sequence without exposure to a high 2,4-D concentration. Considerable variation for callus proliferation was observed. In general, the diploid M. sativa accessions showed poor regenerability and it was not possible to correlate high regeneration frequencies with a particular germplasm source. It was, however, possible to identify regenerable genotypes in all four subspecies. One falcata accession produced somatic embryos on the callus induction media at high frequencies. This response was also obtained with a few genotypes from one xvaria accession. All regenerable plants were maintained as shoot cultures and were able to form somatic embryos on petiole-derived calli. PMID:24253990

A protocol was developed for the induction, maturation and germination of somatic embryos from leaf tissue of jojoba [Simmondsia chinensis (Link) Schneider]. Explants were placed on their adaxial sides in Petri dishes and maintained in darkness on half-strength\\u000a Murashige and Skoog basal medium (MS\\/2). Combinations of 2,4-dichlorophenoxyacetic acid (1.354.52 ?M) with 6-benzylaminopurine\\u000a (1.334.43?M) and 2 synthetic cytokinins, N-(2-chloro-4pyridyl)-N?-phenylurea (1.214.03?M) or

the first report in 1985 (Hakman et al. 1985). Currently, the development of somatic embryos on solid media has been reported in Norway spruce (Picea abies (L. ) Karst) (Hakman et al. 1985; Krogstrup 1986; von Arnold and Hakman 1986), sugar pine (Pinus... slash pine trees (source) were made from an open-pollinated orchard of the Texas Forest Service in Magnolia Springs, Texas during the time interval of June 11, 1991 to July 2, 1991. Cones labeled 1 through 10 correspond to the field identity of each...

A rapid and efficient regeneration system via somaticembryogenesis has been developed from zygotic embryos of Hyoscyamus niger (black henbane). The effect of 2-(n-morpholino)ethanesulfonic acid (MES), myo-inositol (MI) and different combinations of them with a number of growth regulators on somaticembryogenesis was evaluated. Maximum frequency of direct somaticembryogenesis and germination (30.6%) was achieved after 25 weeks by a

Induction of somaticembryogenesis is described in common ash (Fraxinus excelsior L.). Embryogenic tissues are obtained from immature zygotic embryos and cultured on a modified Murashige and Skoog (MS) medium containing 8.8 ?M 2,4-dichlorophenoxyacetic acid and 4.4 ?M benzyl-adenine. Embryogenic tissue is subcultured and multiplied on medium supplemented with reduced concentration of plant growth hormones. Somatic embryos develop and mature by transfer to hormone-free medium and subsequent culture on medium containing low amount of benzyladenine. Somatic embryo germination and conversion are enhanced by cold storage at 4°C and successive transfer onto Woody Plant Medium (WPM). Fully developed plantlets are then transferred to pots and acclimatized in the greenhouse equipped with a mist system. PMID:23179701

Of the various alternatives for cloning elite conifers, somaticembryogenesis (SE) appears to be the best option. In recent years, significant areas of lodgepole pine (Pinus contorta) forest have been devastated by the mountain pine beetle (MPB) in Western Canada. In an attempt to establish an SE propagation system for MPB-resistant lodgepole pine, several families displaying varying levels of resistance were selected for experimentation involving shoot bud and immature seed explants. In bud cultures, eight embryogenic lines were induced from 2 of 15 genotypes following various treatments. Genotype had an important influence on embryogenic culture initiation, and this effect was consistent over time. These lines were identified by microscopic observation and genetic markers. Despite the abundance of early somatic embryos, the cultures have yet to develop into mature embryos. In contrast, immature zygotic embryos (ZEs) cultured from megagametophytes initiated SE at an early dominance stage via nodule-type callus in 1 of 10 genotypes. As part of the study, putative embryogenesis-specific genes, WOX2 (WUSCHELL homeobox 2) and HAP3A, were analyzed in cultures of both shoot bud explants and ZEs. On the basis of these analyses, we postulate that PcHAP3A was expressed mainly in callus and may be involved in cell division, whereas WOX2 was expressed mainly in embryonal mass (EM)-like tissues. The findings from this study, based on molecular assessment, suggest that the cell lines derived from bud cultures were truly EM. Moreover, these experimental observations suggest that PcWOX2 could be used as an early genetic marker to discriminate embryogenic cultures from callus. PMID:20935320

The development of somatic cells in to embryogenic cells occurs in several stages and ends in somatic embryo formation, though most of these biochemical and molecular changes have yet to be elucidated. Somaticembryogenesis coupled with genetic transformation could be a biotechnological tool to improve potential crop yields potential in sugarcane cultivars. The objective of this study was to observe somatic embryo development and to identify differentially expressed proteins in embryogenic (E) and non-embryogenic (NE) callus during maturation treatment. E and NE callus were cultured on maturation culture medium supplemented with different concentrations (0.0, 0.75, 1.5 and 2.0 g L-1) of activated charcoal (AC). Somatic embryo formation and differential protein expression were evaluated at days 0 and 21 using shotgun proteomic analyses. Treatment with 1.5 g L-1 AC resulted in higher somatic embryo maturation rates (158 somatic embryos in 14 days) in E callus but has no effect in NE callus. A total of 752 co-expressed proteins were identified through the SUCEST (The Sugarcane EST Project), including many housekeeping proteins. E callus showed 65 exclusive proteins on day 0, including dehydrogenase, desiccation-related protein, callose synthase 1 and nitric oxide synthase. After 21 days on maturation treatment, 14 exclusive proteins were identified in E callus, including catalase and secreted protein. NE callus showed 23 exclusive proteins on day 0 and 10 exclusive proteins after 21 days on maturation treatment, including many proteins related to protein degradation. The induction of maturation leads to somatic embryo development, which likely depends on the expression of specific proteins throughout the process, as seen in E callus under maturation treatment. On the other hand, some exclusive proteins can also specifically prevent of somatic embryos development, as seen in the NE callus. PMID:26035435

The development of somatic cells in to embryogenic cells occurs in several stages and ends in somatic embryo formation, though most of these biochemical and molecular changes have yet to be elucidated. Somaticembryogenesis coupled with genetic transformation could be a biotechnological tool to improve potential crop yields potential in sugarcane cultivars. The objective of this study was to observe somatic embryo development and to identify differentially expressed proteins in embryogenic (E) and non-embryogenic (NE) callus during maturation treatment. E and NE callus were cultured on maturation culture medium supplemented with different concentrations (0.0, 0.75, 1.5 and 2.0 g L-1) of activated charcoal (AC). Somatic embryo formation and differential protein expression were evaluated at days 0 and 21 using shotgun proteomic analyses. Treatment with 1.5 g L-1 AC resulted in higher somatic embryo maturation rates (158 somatic embryos in 14 days) in E callus but has no effect in NE callus. A total of 752 co-expressed proteins were identified through the SUCEST (The Sugarcane EST Project), including many housekeeping proteins. E callus showed 65 exclusive proteins on day 0, including dehydrogenase, desiccation-related protein, callose synthase 1 and nitric oxide synthase. After 21 days on maturation treatment, 14 exclusive proteins were identified in E callus, including catalase and secreted protein. NE callus showed 23 exclusive proteins on day 0 and 10 exclusive proteins after 21 days on maturation treatment, including many proteins related to protein degradation. The induction of maturation leads to somatic embryo development, which likely depends on the expression of specific proteins throughout the process, as seen in E callus under maturation treatment. On the other hand, some exclusive proteins can also specifically prevent of somatic embryos development, as seen in the NE callus. PMID:26035435

The focus of the current project was to establish somaticembryogenesis protocols for the tropical pine species Pinus oocarpa using immature zygotic embryos (ZEs) as explants. Somaticembryogenesis is best supported by mimicking natural seed-embryo developmental conditions, through a tissue culture medium formulation based on the mineral content of the seed nutritive tissue [megagametophyte (MG)]. A novel culture medium (P. oocarpa medium, PO) was tested in combination with different plant growth regulator (PGR) concentrations and compared with standard Pinus taeda media for the initiation of somaticembryogenesis from immature ZEs of P. oocarpa. Immature MGs containing immature ZEs of two mother trees were used with 12 and 8% extrusion rates for mother tree genotypes 3 and 5, respectively. In both mother trees the percentage capture was 2%. Multiplication of two captured cell lines (T5C2S01 and T5C1S12) was improved by lowering the concentrations of PGRs to 2.5&emsp14;µM each 2,4-dichlorophenoxyacetic acid and abscisic acid (ABA) plus 1.0&emsp14;µM each 6-benzylaminopurine and kinetin. Mature somatic embryos formed on 40&emsp14;µM ABA, 6% (w/v) maltose, 12% (w/v) PEG 8000 and 0.6% (w/v) Phytagel. While PO medium appeared suboptimal for somatic embryo induction, it did exhibit potential for enhanced culture proliferation and subsequent improved maturation with cell line T5C2S01, where microscopic analysis revealed better embryo morphology on PO medium than on 1250 medium. However, this enhancement was not observed with cell line T5C1S12. Germination was preceded by partial desiccation for a period of 2-3 weeks before transferring the embryos to germination medium. Germination was observed after 7 days under low light, and apical primordia slowly expanded after transfer to ex vitro conditions. To our knowledge, this is the first report on the production of somatic seedlings in P. oocarpa. PMID:21636694

Podophyllum hexandrum Royle known as Indian mayapple is an important medicinal plant found only in higher altitudes (2,700 to 4,200 m) of the Himalayas. The highly valued anticancer drug Podophyllotoxin is obtained from the roots of this plant. Due to over exploitation, this endemic plant species is on the verge of extinction. In vitro culture for efficient regeneration and the production of podophyllotoxin is an important research priority for this plant. Hence, in the present study, an efficient plant regeneration system for mass multiplication through somaticembryogenesis was developed. We have screened P. hexandrum seeds collected from three different regions in the Himalayas to find their regenerative potentials. These variants showed variation in germination percentage as well as somatic embryogenic frequency. The seeds collected from the Milam area of Pithoragarh district showed better germination response (99.3%) on Murashige and Skoog (MS) medium fortified with Gibberellic acid (GA3 [5 mg/l]) and higher direct somatic embryogenic frequency (89.6%). Maximum production of embryogenic callus (1.2 g fresh weight [FW]) was obtained when cotyledons containing the direct somatic embryo clusters were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D [1.5 mg/l]) after 4 week of culture in complete darkness. In the present investigation, somaticembryogenesis was accomplished either by direct organogenesis or callus mediated pathways. The latter method resulted in a higher frequency of somatic embryo induction in hormone-free MS medium yielding 47.7 embryos/50 mg of embryogenic callus and subsequent germination in MS medium supplemented with GA3 (5 mg/l). Seventy-nine percent of embryos attained complete maturity and germinated into normal plants with well-developed roots. Systematic histological analysis revealed the origin of somatic embryo and their ontogenesis. The higher level of podophyllotoxin (1.8 mg/g dry weight [DW]) was recorded in germinated somatic embryos when compared to field grown plants. The present system can be widely used for mass propagation, transgenic recovery, and podophyllotoxin production for commercial utilization. PMID:24633328

Nickel (0.13, 0.25, 0.5, 1.0 and 1.5 mg\\/l) increased the efficiency of somaticembryogenesis in leaf base and mesocotyl derived\\u000a calli of Setaria italica. A lower concentration of nickel in the culture media promoted long-term maintenance of embryogenic\\u000a calli that regenerated into plantlets. The plants obtained from embryogenic calli grown on Ni-containing medium showed tolerance\\u000a to nickel. The growth of

Immature embryos of sawtooth oak (Quercus acutissima Carruth.) were obtained from five seed families and cultured on modified Murashige and Skoog nutrient medium containing 1\\u000a g\\/l l-glutamine and 5 mM proline and supplemented with 1.0 mg\\/l indole-3-butyric acid and 1.0 mg\\/l 6-benzylaminopurine. The frequency of somaticembryogenesis\\u000a from immature embryos was a function of the collection date and seed family.

Most of the somaticembryogenesis (SE) process requires the presence, either before or during the embryogenic process, of at least one exogenous auxin. This exogenous auxin induces the presence of endogenous auxins, which appears to be essential for SE induction. We found that during the preincubation period of SE in Coffea canephora, there is an important increase in both free and conjugated indole-3-acetic acid (IAA), as well as indole-3-butyric acid. This increase is accompanied by an increase in the expression of YUCCA (CcYUC), TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (CcTAA1), and GRETCHEN HAGEN 3 (GH3) genes. On the other hand, most of the IAA compounds decreased during the induction of SE. The results presented in this research suggest that a balance between free IAA and its amide conjugates is necessary to allow the expression of SE-related genes. PMID:24299659

.Using immature zygotic embryos as explants, we have developed an efficient method for somaticembryogenesis in three germplasm accessions collected from China, India and Indonesia. Indirect somaticembryogenesis was achieved when endosperm tissue and immature embryos between 0.5-1.0 cm in length were cultured in a medium with 2,4-D, preferably at 5-10 mg/l, followed by a shift to a hormone-free medium supplemented with glutamine and asparagine. Production of secondary embryos was improved by supplementing KNO3, glutamine and asparagine. 2,4-D (0.1-0.2 mg/l). PEG 8000 (5-10%) were essential for maintenance of embryogenic calli in liquid medium. Regeneration of soil-ready plants took as short as 3 months using the suspension cultures. Over 95% of the regenerated trees were able to flower and set seeds with no discernable morphological abnormality. This regeneration method is expected to facilitate the development of more efficient transformation system for Jatropha curcas. PMID:21865864

A simple and efficient protocol was developed for somaticembryogenesis from leaf and petiole explants of Campanula punctata Lam. var. rubriflora Makino. Somatic embryos (SE) were obtained with greater frequency from petiole explants than from leaf explants when cultured\\u000a on Murashige and Skoog (MS) medium supplemented with 2.0 mg L?1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L?1 6-benzyladenine (BA). On this medium, a mean

To optimize an in vitro protocol for propagation of saffron through somaticembryogenesis, effects of various concentrations of 2,4-D ( 0, 0.25, 0.5, 1, 2, 4 and 8 mg L(-1)) in combination with BAP (0, 0.25, 0.5, 1, 2, 4 and 8 mg L(-1)) were studied. Surface-sterilized corms were cut transversally into equal portions and the upper or lower parts were used separately as explants. All treatments were maintained in the darkness at 24 +/- 2 degrees C. After 70 days, the first globular embryos were observed and the number of embryos on each explant reached to its maximum 3 months after culture. Statistical analysis showed that there were significant differences between treatments regarding the number of embryos induced on each explant. The most effective treatment was 2.0 mg L(-1) 2,4-D + 1.0 mg L(-1) BAP for both types of explant (inducing 6.5 +/- 1.3 and 35.95 +/- 4.9 embryos on each explant for the upper and lower parts, respectively). The average percentages of explants showing embryogenic response were 33.3 and 93.3% for the upper and the lower part of corm tissue respectively in this treatment. Complementary studies are in progress to optimize maturation and germination stages of these somatic embryos. PMID:19090256

The number of cortical microtubules (MTs) increases considerably as cultured carrot (Daucus carota L.) cells initiate and progress through somaticembryogenesis. The basis for this increase in MT number was investigated. A radioimmune assay was used to show that tubulin-protein per cell first decreased as the undifferentiated cells initiated embryonic development, but subsequently increased approximately fivefold between the globular and

Plantlets were obtained from leaf explants of a Labiatae tree - Leucosceptrum canum Sm. using plant tissue culture techniques. Two types of calli proliferated from the leaf explants when grown on different media, one of which was amenable to somaticembryogenesis. Differentiation of the embryoids started from the fourth passage of culture and continued up to the seventh passage. The number of embryoids decreased with the age of the callus. The capacity of such embryoids to form entire plantlets was studied using different nutrient mileux. Embryoids formed plantlets on Murashige and Skoog's (MS) medium fortified with benzylaminopurine plus indolebutyric acid. Organogenesis was observed in shoot-buds derived from explants of in vitro regenerated plantlets on MS basal medium supplemented with benzylaminopurine. Culture regenerated plantlets were transferred to MS medium without sucrose and growth hormones; finally transferred to pots containing sterile vermiculite where they are growing. PMID:24253989

Triticum aestivum is of major importance both nutritionally and economically. Introduction of new genes has been difficult to apply to elite wheat varieties mainly as a result of their recalcitrance to prerequisite tissue culture. We attempted to improve the frequency of wheat transformation by exposing plants to high level of ammonium nitrate. Our experiments showed that modification of the ammonium nitrate content in the direct somaticembryogenesis induction medium can increase the number of primary embryos produced over twofold in the elite hard red wheat cultivar Superb. The number of primary embryos that were capable of transitioning into shoot development also increased twofold. Biolistic transformation efficiency improved as much as sevenfold when targeted scutellar tissue was exposed to elevated ammonium nitrate levels. This simple approach could become extremely useful for increasing transformation efficiency in wheat. PMID:19818315

Development of highly efficient and reproducible plant regeneration system has tremendous potential to provide improved technology to assist in genetic transformation of indica rice cultivars for their further exploitation in selection. For the development of a highly reproducible regeneration system through somaticembryogenesis, mature embryos of highly popular rice cultivars i.e., Govind (for rainfed areas), Pusa Basmati-1 (aromatic basmati) and Jaya (for irrigated areas) were used. Optimum callus formation (%) to MS medium supplemented with 2, 4-D was obtained at 12.0 microM in Govind, 14.0 microM in Jaya and 15.0 microM in Pusa Basmati-1. All the cultivars showed good proliferation on MS medium without hormone. In Govind, highest embryogenic response was observed in MS medium supplemented with 2, 4-D (0.4 microM) + kinetin (0.4 microM), while in Pusa Basmati-1 with 2, 4-D (0.4 microM) + kinetin (2.0 microM) and in Jaya on hormone-free MS medium. Excellent embryo regeneration in Govind was observed on MS medium supplemented with low concentrations (1.1 microM) of BAP or hormone-free MS medium, while in Pusa Basmati-1 and Jaya embryogenesis was observed on MS medium supplemented with higher concentration of BAP (2.2 microM). Similarly, maximum plantlets with proliferated roots were observed in Govind on hormone-free MS medium, while in Pusa Basmati-1 and Jaya on MS medium supplemented with high concentration of NAA (4.0 microM). Developed plantlets were further successfully acclimatized and grown under pot culture up to maturity. Further the yield potential of in vitro developed plants was accessed at par to the direct seeded one under pot culture. Present, protocol standardizes somaticembryogenesis and efficient regeneration of agronomically important, high yielding and diverse indica rice cultivars which can be utilized as an efficient tool for molecular studies and genetic transformation in future. PMID:22403871

The purpose of the study was to evaluate the possible genetic effect on vegetative propagation of Coffea canephora. Diversity for somaticembryogenesis (SE) ability was observed not only among two groups of C. canephora Pierre (Congolese and Guinean), but also within these different genetic groups. The results therefore showed that, under given experimental conditions, SE ability is depending on genotype. Furthermore the detection of quantitative trait loci (QTLs) controlling the SE and cutting abilities of C. canephora was performed on a large number of clones including accessions from a core collection, three parental clones and their segregating progenies. On the one hand we detected eight QTLs determining SE. Six positive QTLs for SE ability, whatever the criteria used to quantify this ability, were localized on one single chromosome region of the consensus genetic map. Two negative QTLs for SE ability (frequency of micro calli without somatic embryo) were detected on another linkage group. Deep analysis of the six QTLs detected for SE ability came to the conclusion that they can be assimilated to one single QTL explaining 8.6-12.2% of the observed variation. On the other hand, two QTLs for average length of roots and length of the longest sprouts of cuttings were detected in two linkage groups. These QTLs detected for cutting ability are explaining 12-27% of the observed variation. These observations led to conclude that SE and cutting abilities of C. canephora Pierre appeared to be genetic dependent but through independent mechanisms. PMID:20145933

The degree of genetic control and the effects of cultural treatments on somaticembryogenesis (SE) in white spruce were investigated with material derived from six-parent diallel crosses, including reciprocals. Thirty zygotic embryos from both immature and mature cones of each family were cultured in media with either 2,4-D or Picloram immediately after the collection of cones and after 2 months

We have developed a reliable and high-frequency system of transformation and regeneration via somaticembryogenesis (SE) of Lycium barbarum. Leaf segments were co-cultivated with Agrobacterium tumefaciens EHA101 (pIG121Hm) carrying the neomycin phosphotransferase II gene as a selectable marker and an intron-#-glucuronidase (GUS) gene as a reporter marker. On the medium for callus-induction, which contained 50 mg l-1 kanamycin (Km), approximately

Summary The most important advantage of cloning conifers by somaticembryogenesis (SE) is that the embryogenic tissue can be cryopreserved\\u000a without changing its genetic make-up and without loss of juvenility. This offers an opportunity to develop high-value clonal\\u000a varieties by defrosting and repropagating cryopreserved clones after genetic testing has shown which clones are the best performers.\\u000a In the current absence of

Basal media and plant growth regulators were tested for the promotion of somaticembryogenesis from immature wheat-rye hybrid embryos. Influence of growth regulators and chilling on plant regeneration were tested on two media. A medium containing four amino acids-glutamine, arginine, glycine and aspartic acid-as the nitrogen source, promoted the production of, on average, twice as much embryogenic callus as the

Calli were induced from the crown of seedlings or lateral bud of young spears of Asparagus officinalis L. on Linsmaier and Skoog's (LS) solid-medium supplemented with 5 µ M 2,4-dichlorophenoxyacetic acid (2,4-D). Embryogenic callus was selected from induced calli and proliferated in LS liquid medium supplemented with 5 µ M 2,4-D. Non-vitrified somatic embryos were formed and efficiently developed into

Somaticembryogenesis (SE) in Cichorium involves dedifferentiation and redifferentiation of single cells and can be induced by specific in vitro culture conditions. We have tested the effect of various treatments on the incidence of SE (ISE) of an interspecific embryogenic hybrid (C. endivia x C. intybus) and of different commercial chicories (C. endivia and C. intybus) that are typically recalcitrant to SE in standard culture conditions. We found that the ISE of the hybrid is significantly increased by pretreatment of tissues by submersion in solutions of glycerol, abscisic acid, spermine, putrescine or of combinations of these compounds. Interestingly, the most efficient of these pretreatments also had an unexpectedly high effect on the ISE of the C. intybus cultivars. The ISE of the hybrid and of the commercial chicories were increased when explants were co-cultured with highly embryogenic chicory explants or when they were cultured in conditioned medium. These observations established that unidentified SE-promoting factors are released in the culture medium. HPLC analyses of secreted Arabino-Galactan Proteins (AGPs), which are known to stimulate SE, did not allow identifying a fraction containing differentially abundant AGP candidates. However, pointing to their role in promoting SE, we found that the hybrid had a drastically higher ISE when amino sugars and L-Proline, the putative precursors of secreted AGPs, were both added to the medium. PMID:22301978

Glutathione S-tranferases (GSTs) are a heterogeneous family of proteins, which perform diverse pivotal catalytic and non-enzymatic functions during plant development and in plant stress responses. Previous studies have shown that a GST activity (EC 2.5.1.18) is closely linked with the precocious phases of somaticembryogenesis in leaf tissues of an interspecific chicory hybrid (Cichorium intybus L. var. sativa × C. endivia L. var. latifolia). In order to learn more about the involvement of this enzyme in this process, in situ-hybridization as well as immunolocalization were performed in parallel. GST-mRNAs and proteins were colocalized in small veins, particularly in young protoxylem cell walls. During cell reactivation, the in situ and protein signals became less intense and were associated with chloroplasts. The GST-mRNAs and corresponding proteins were not always colocalized in the same tissues. While high amounts of transcripts could be detected in multicellular embryos, the proteins were not well labeled. Our results indicated that GSTs belong to a complex anti-oxidant mechanism within the cell, and also at the cell wall level. GSTs presence in reactivated cell and multicellular embryos is discussed in relation to redox cell status. PMID:19516999

Little is known about the genes expressed during grapevine somaticembryogenesis. Both groups of SomaticEmbryogenesis Receptor Kinase (SERK) and Leafy Cotyledon (LEC and L1L) genes seem to play key roles during somaticembryogenesis in various plant species. Therefore, we identified and analysed\\u000a the sequences of VvSERK and VvL1L (Leafy cotyledon1-Like) genes. The deduced amino acid sequences of VvSERK1, VvSERK2

The frequency of long-term secondary somaticembryogenesis and shoot meristem development from embryogenic masses of the cherry rootstock `Colt' ( Prunus avium × P. pseudocerasus), differentiated from transgenic roots containing the T-DNA of Agrobacterium rhizogenes, has opened the way for genetic improvement by biotechnological techniques. Whole plants were produced by stimulating shoot meristem development from somatic embryos. The combination of

Globular somatic embryos can be induced from immature cotyledons of soybean (Glycine max L. Merr. cv Jack) placed on high levels of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos develop from the adaxial side of the cotyledon, whereas the abaxial side evolves into a callus. Using a 9,280-cDNA clone array, we have compared steady-state RNA from the adaxial side from which embryos develop and from the abaxial callus at five time points over the course of the 4 weeks necessary for the development of globular embryos. In a second set of experiments, we have profiled the expression of each clone in the adaxial side during the same period. A total of 495 genes differentially expressed in at least one of these experiments were grouped according to the similarity of their expression profiles using a nonhierarchical clustering algorithm. Our results indicate that the appearance of somatic embryos is preceded by dedifferentiation of the cotyledon during the first 2 weeks on auxin. Changes in mRNA abundance of genes characteristic of oxidative stress and genes indicative of cell division in the adaxial side of the cotyledons suggest that the arrangement of the new cells into organized structures might depend on a genetically controlled balance between cell proliferation and cell death. Our data also suggest that the formation of somatic globular embryos is accompanied by the transcription of storage proteins and the synthesis of gibberellic acid. PMID:12746518

A new micropropagation system for Lycium barbarum (L.) was developed using root explants as starting material. Callus can be produced from root explants on Murashige and Skoog\\u000a (MS) medium containing 0.2 mg dm?3 2,4-dichlorophenoxyacetic acid. After three subcultures on the same medium, callus was then transferred onto the MS medium\\u000a supplemented with 500 mg dm?3 lactalbumin hydrolysate to inducesomatic

Somatic embryos induced from callus cultures of lemongrass [Cymbopogon flexuosus (Nees) Wats.] on Murashige and Skoog medium supplemented with 5 mg\\/l of 2,4-D, 0.1 mg\\/l of NAA and 0.5 mg\\/l of Kn developed into plantlets when plated on a medium supplemented with 3 mg\\/l of BA, 1 mg\\/l of GA3 and 0.1 mg\\/l of NAA. The regeneration potential of callus

An efficient and reproducible procedure is established for the plant regeneration from hypocotyl explants and hypocotyl-or stem-derived calli in Astragalus melilotoides. High frequency somatic embryo formation (98.3%) occurred direct on hypocotyls on Murashige and Skoog (MS) medium supplemented with 2.69 µM NAA and 4.44 µM BA within 5 weeks. Three types of calli were induced from the hypocotyl and stem

Immature and mature nonstratified seeds of white ash (Fraxinus americana L.) were dissected transversely and 2\\/3 of each seed was placed onto agar-solidified Murashige and Skoog medium. Adventitious buds, shoots, and somatic embryos formed on callus, cotyledons, and hypocotyls of the resulting seedlings. Shoot organogenesis was induced on explants cultured on medium with 10 µM thidiazuron but not on explants

MicroRNAs (miRNAs) are endogenous non-coding ~21 nucleotide RNAs that regulate gene expression at the transcriptional and post-transcriptional levels in plants and animals. They play an important role in development, abiotic stress, and pathogen responses. miRNAs with their targets have been widely studied in model plants, but limited knowledge is available on the small RNA population of cotton (Gossypium hirsutum)an important economic crop, and global identification of related targets through degradome sequencing has not been developed previously. In this study, small RNAs and their targets were identified during cotton somaticembryogenesis (SE) through high-throughput small RNA and degradome sequencing, comparing seedling hypocotyl and embryogenic callus (EC) of G. hirsutum YZ1. A total of 36 known miRNA families were found to be differentially expressed, of which 19 miRNA families were represented by 29 precursors. Twenty-five novel miRNAs were identified. A total of 234 transcripts in EC and 322 transcripts in control (CK) were found to be the targets of 23 and 30 known miRNA families, respectively, and 16 transcripts were targeted by eight novel miRNAs. Interestingly, four trans-acting small interfering RNAs (tas3-siRNAs) were also found in degradome libraries, three of which perfectly matched their precursors. Several targets were further validated via RNA ligase-mediated rapid amplification of 5 cDNA ends (RLM 5-RACE). The profiling of the miRNAs and their target genes provides new information on the miRNAs network during cotton SE. PMID:23382553

In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and

We report here a method for plant regeneration through somaticembryogenesis from explants collected from immature male inflorescence of adult oil palm cultivated in India. Callus induction was successful from tissues of immature male inflorescence collected from both dura and tenera varieties of oil palm. A modified Y3 (Eeuwens) media supplemented with several additives and activated charcoal (3%) were used for the experiments. Out of four different auxin treatments, 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid (picloram) produced maximum callus induction (82%) and it was not significantly different from 2,4-dichlorophenoxyacetic acid (2,4-D) and a combination of 2,4-D + picloram. The callus induction obtained with auxin ?-naphthalene acetic acid was only 54% and it was significantly low as compared to the other treatments. Highest embryogenesis was obtained with a combination of 2,4-D + picloram (4.9%) followed by picloram (3.4%). Genotypic variation in response to the same auxins was observed both for callus induction and embryogenesis. Callus induction and embryogenesis ranged from 42 to 72% and 6.8 to 9.35%, respectively in tenera. The formation of embryogenic calli was marked by the appearance of white to yellowish globular or nodular structures which subsequently formed clear somatic embryos. Somaticembryogenesis was asynchronous and at one time we could find different stages of embryogenesis like the globular, torpedo and the cotyledonary stages. The somatic embryos when exposed to light in the same basal media along with 6-benzyladenine (18 µM), abscisic acid (3.78 µM) and gibberellic acid (5.78 µM) regenerated into plantlets. To the best of our knowledge this is the first report o f callus induction and somaticembryogenesis from immature male inflorescence of oil palm. PMID:26085976

Somaticembryogenesis (SE) is a poorly understood process during which competent cells respond to inducing conditions, allowing the development of somatic embryos. It is important for the regeneration of transgenic plants, including for soybean (Glycine max). We report here that constitutive expression of soybean orthologs of the Arabidopsis (Arabidopsis thaliana) MADS box genes Agamous-like15 (GmAGL15) and GmAGL18 increased embryogenic competence of explants from these transgenic soybean plants. To understand how GmAGL15 promotes SE, expression studies were performed. Particular genes of interest involved in embryogenesis (abscisic acid-insensitive3 and FUSCA3) were found to be directly up-regulated by GmAGL15 by using a combination of quantitative reverse transcription-polymerase chain reaction and chromatin immunoprecipitation. To look more broadly at changes in gene expression in response to GmAGL15, we assessed the transcriptome using the Affymetrix Soybean Genome Array. Interestingly, the gene expression profile of 35Spro:GmAGL15 explants (0 d in culture) was found to resemble nontransgenic tissue that had been induced for SE by being placed on induction medium for 3 d, possibly explaining the more rapid SE development observed on 35Spro:GmAGL15 tissue. In particular, transcripts from genes related to the stress response showed increased transcript accumulation in explants from 35Spro:GmAGL15 tissue. These same genes also showed increased transcript accumulation in response to culturing nontransgenic soybean explants on the medium used to induce SE. Overexpression of GmAGL15 may enhance SE by making the tissue more competent to respond to 2,4-dichlorophenoxyacetic acid induction by differential regulation of genes such as those involved in the stress response, resulting in more rapid and prolific SE. PMID:24481137

Somatic embryos were obtained and plants regenerated from immature embryos of onion following culture on embryogenic induction\\u000a media. Highest rates of somatic embrogenesis resulted from 0.5- to 1.5-mm immature embryos cultured on media containing 5\\u000a mg\\/l of picloram. Somatic embryos formed either directly on the surface of embryos or developed from compact cultures. The\\u000a production of somatic embryos was significantly

Somaticembryogenesis (SE) is a remarkable process of plant somatic cells developing into an embryo capable of forming a complete\\u000a plant. MiRNAs play important roles in plant development by regulating expression of their target genes, but its function in\\u000a SE has rarely been studied. Herein, ten conserved miRNAs with critical functions in plant development are detected by stem-loop\\u000a qRT-PCR in

Background Hydroxyproline rich glycoproteins (HRGPs) are implicated to have a role in many aspects of plant growth and development but there is limited knowledge about their localization and function during somaticembryogenesis of higher plants. In this study, the localization and function of hydroxyproline rich glycoproteins in embryogenic cells (ECs) and somatic embryos of banana were investigated by using immunobloting and immunocytochemistry with monoclonal JIM11 and JIM20 antibodies as well as by treatment with 3,4-dehydro-L-proline (3,4-DHP, an inhibitor of extensin biosynthesis), and by immunomodulation with the JIM11 antibody. Results Immunofluorescence labelling of JIM11 and JIM20 hydroxyproline rich glycoprotein epitopes was relatively weak in non-embryogenic cells (NECs), mainly on the edge of small cell aggregates. On the other hand, hydroxyproline rich glycoprotein epitopes were found to be enriched in early embryogenic cells as well as in various developmental stages of somatic embryos. Embryogenic cells (ECs), proembryos and globular embryos showed strong labelling of hydroxyproline rich glycoprotein epitopes, especially in their cell walls and outer surface layer, so-called extracellular matrix (ECM). This hydroxyproline rich glycoprotein signal at embryo surfaces decreased and/or fully disappeared during later developmental stages (e.g. pear-shaped and cotyledonary stages) of embryos. In these later developmental embryogenic stages, however, new prominent hydroxyproline rich glycoprotein labelling appeared in tri-cellular junctions among parenchymatic cells inside these embryos. Overall immunofluorescence labelling of late stage embryos with JIM20 antibody was weaker than that of JIM11. Western blot analysis supported the above immunolocalization data. The treatment with 3,4-DHP inhibited the development of embryogenic cells and decreased the rate of embryo germination. Embryo-like structures, which developed after 3,4-DHP treatment showed aberrant non-compact epidermis with discontinuous ECM at the outer surface as well as much less immunolabelling with the JIM11 antibody. This treatment also decreased the plant regeneration capacity in embryogenic banana cultures. Finally, immunomodulation of surface hydroxyproline rich glycoproteins by co-culture of embryos with the JIM11 antibody resulted in a much lower germination capacity of these embryos. Conclusions These results suggest that hydroxyproline rich glycoproteins play an important developmental role, especially in the process of regeneration and germination of embryos during plant regeneration via somaticembryogenesis. Proper content and localization of hydroxyproline rich glycoproteins seem to be essential for the formation and regeneration of banana somatic embryos. PMID:21349190

Irones are violet-scented ketonic compounds contained in the rhizome of certain species of iris. As cultivation of the iris tends to decrease, a selection program has been initiated to find the best performing clones in terms of growth and yield. Parallel to this selection, in vitro regeneration studies have been carried out in order to multiply interesting clones. A method of rapid multiplication by somaticembryogenesis associated with multibudding was developed. Callus was obtained from leaf bases, flower pieces or rhizome apices; the best explants were flower pieces. The induction media used to obtain embryogenic callus were Murashige & Skoog (1962) media. Assays with adding of proline in these media have showed that it could double the yield of embryogenic callus. The embryogenic expression medium was the Knudson's orchid agar (Knudson 1946) medium. Conformity of the plants obtained was checked by comparing their chemotypes with those of the mother plants. PMID:24193517

We report here the isolation of the Arabidopsis SOMATICEMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (AtSERK1) gene and we demonstrate its role during establishment of somaticembryogenesis in culture. The AtSERK1 gene is highly expressed during embryogenic cell formation in culture and during early embryogenesis. The AtSERK1 gene is first expressed in planta during megasporogenesis in the nucleus of developing ovules, in the functional megaspore, and in all cells of the embryo sac up to fertilization. After fertilization, AtSERK1 expression is seen in all cells of the developing embryo until the heart stage. After this stage, AtSERK1 expression is no longer detectable in the embryo or in any part of the developing seed. Low expression is detected in adult vascular tissue. Ectopic expression of the full-length AtSERK1 cDNA under the control of the cauliflower mosaic virus 35S promoter did not result in any altered plant phenotype. However, seedlings that overexpressed the AtSERK1 mRNA exhibited a 3- to 4-fold increase in efficiency for initiation of somaticembryogenesis. Thus, an increased AtSERK1 level is sufficient to confer embryogenic competence in culture. PMID:11706164

Background and Aims In Tilia amurensis, two types of trichomes (hairy and glandular) develop from epidermal surfaces of cotyledons and hypocotyls of zygotic embryos soon after germination. Here, it is demonstrated that glandular trichome initials develop directly into somatic embryos when treated in vitro with 2,4-dichlorophenoxyacetic acid (2,4-D). Methods Zygotic embryos of Tilia amurensis were cultured on Murashige and Skoog medium with 3 % sucrose and various concentrations (0, 2·2, 4·4 and 8·8 µm) of 2,4-D. Morphological development of trichomes and somatic embryos was analysed by scanning electron microscope and light microscope after histological sectioning. Key Results In zygotic embryos cultured on medium with 4·4 µm 2,4-D, formation of hairy trichomes was completely suppressed but formation of glandular trichome initials increased. That some filamentous trichome initials developed directly into somatic embryos was confirmed by histological and scanning electron microscope observation. When explants with different stages of trichome initials (two-, four- and eight-celled filamentous and fully mature trichomes) were temporally pre-treated with 4·4 µm 2,4-D for 24 h and transferred into hormone-free medium, two-celled and four-celled filamentous trichome initials were the effective stage of trichomes for somatic embryo induction. Conclusions It is suggested that early developing filamentous trichome initials have developmental plasticity and that with 2,4-D treatment these trichome initials develop directly into somatic embryos. PMID:17565972

Effects of application in vivo of glyphosate, fluridone, and paclobutrazol to glasshouse-grown donor plants of Pennisetum purpureum Schum. on endogenous levels of abscisic acid (ABA) and indole-3-acetic acid (IAA) in young leaves and on somaticembryogenesis in cultured leaf explants were studied. Treatment of plants with glyphosate (100 milligrams per liter) resulted in elevated levels of endogenous ABA and IAA in young leaves. In contrast, paclobutrazol (50% active ingredient; 200 milligrams per liter) did not alter the endogenous levels of ABA and IAA. Fluridone (100 milligrams per liter) markedly inhibited synthesis of ABA and leaf explants from fluridone-treated plants lost the capacity for somaticembryogenesis. Explants from glyphosate- or paclobutrazol-treated plants did not show any reduction in embryogenic capacity when compared with untreated control plants. Glyphosate and fluridone were also incorporated into the culture media at various concentrations (0 to 20 milligrams per liter) to study their effects in vitro on somaticembryogenesis in leaf explants from untreated, field-grown plants. Glyphosate was inhibitory to somaticembryogenesis but only at concentrations above 5 milligrams per liter. Fluridone inhibited somaticembryogenesis at all concentrations tested. Inhibition of somaticembryogenesis by fluridone, by either in vivo or in vitro application, could be overcome partially by (+/-)-ABA added to the culture medium. Exogenous application of (+/-)-ABA enhanced somaticembryogenesis and reduced the formation of nonembryogenic callus. Application of IAA or gibberellic acid (GA(3); >5 milligrams per liter) was inhibitory to somaticembryogenesis. These results indicate that endogenous ABA is one of the important factors controlling the embryogenic capacity of leaf explants in Napier grass. PMID:16665403

Plant regeneration of buffalograss `Texoka' was achieved through both somaticembryogenesis and organogenesis by culturing immature male inflorescences collected from field-grown plants. Three passages of subculture for calluses derived from male `Texoka' on medium containing 2.25, 4.5, or 9 µM 2,4-D combined with either 0.44 µM or 1.32 µM BA led to shoot formation via organogenesis. Higher concentrations of 2,4-D

The most important commercial species of coffee, Coffea arabica, which produces 73% of the world's coffee crop and almost all of the coffee in Latin America, is the only tetraploid (allotetraploid,\\u000a 2n=4x=44) species known in the genus. High-frequency somaticembryogenesis, plant regeneration and plant recovery were achieved\\u000a from leaf explants of a mature, elite plant of C. arabica cv. Cauvery

The effects of different factors on the embryogenesis and plant regeneration from mature embryos of Russian spring and winter\\u000a genotypes were studied. Embryogenic callus induction was achieved on MS medium supplemented with different concentrations\\u000a of 2,4-D (2,4-dichlorophenoxyacetic acid), 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) or Dicamba (3,6-dichloro-o-anisic acid). Although all auxins were able to induce callus from explants with high frequency (98100%), Dicamba

The effect of boric acid on somatic embryo induction in rice was investigated using mature seeds of a widely used cytoplasmic\\u000a male sterile line of indica rice IR-58025. Boric acid was added at a concentration of 100.00, 161.29, 241.93, 322.58 and 403.22\\u000a ?M to the embryo induction medium containing basal salts, with 9.84 ?M 2,4-dichlorophenoxyacetic acid. Boric acid was found

Interactions of varying ancymidol concentrations with those of ?-naphthaleneacetic acid (NAA) or sucrose in embryo induction medium were related to the production and development of asparagus\\u000a (Asparagus officinalis L.) somatic embryos, and to the ability of these embryos to germinate. A significant sucrose×ancymidol interaction was observed\\u000a only for the production of bipolar embryos; 4% sucrose with 0.75 mg l1 ancymidol

The effects of O2, growth-regulators and desiccation on callus growth and somatic embryo (embryoid) development were investigated in cultures of immature embryos of two lines of Triticum aestivum L. Callus and embryoid formation were induced on media that contained N6-furfurylamin-opurine (kinetin) and either 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-o-anisic acid, either with or without abscisic acid (ABA). Cultures containing differentiated embryoids were

Adult conifers are still recalcitrant in clonal propagation despite significant advances in forest tree biotechnology. Plant regeneration through somaticembryogenesis from explants older than mature zygotic embryos is either difficult or impossible to achieve. To investigate if ectopic expression of transcription factors involved in the induction of the embryogenic process would inducesomaticembryogenesis in Picea glauca (white spruce) somatic plants, we used the LEAFY-COTYLEDON1 homolog cloned from Picea mariana, CHAP3A, and Arabidopsis thaliana WUS to transform embryonal mass of P. glauca. Ectopic gene expression was induced by 17-beta-estradiol during stages of somaticembryogenesis (early embryogenesis and late embryogenesis) and somatic seedling growth in the transgenics. Of the two transcription factors, only WUS produced severe phenotypes by disrupting the development of somatic embryos on the maturation medium and inhibiting germination. However, none of the transgenes induced ectopic somaticembryogenesis even in the presence of plant growth regulators. Absolute quantitative PCR confirmed the expression of both CHAP3A and WUS in transgenic embryonal mass and in all parts of somatic seedlings. A high expression of the transgenes did not influence expression profiles of any of the ten other transcription factors tested, some of which have been known to be involved in the process of embryogenesis. Implications of these results for further work are discussed. PMID:20424847

A reproducible procedure for induction of somaticembryogenesis (SE) from adult trees of Eucalyptus globulus Labill. and the hybrid E. saligna Smith?×?E. maidenii has been developed for the first time. Somatic embryos were obtained from both shoot apex and leaf explants of all three genotypes evaluated, although embryogenic frequencies were significantly influenced by the species/genotype, auxin and explant type. Picloram was more efficient for somatic embryo induction than naphthaleneacetic acid (NAA), with the highest frequency of induction being obtained in Murashige and Skoog medium containing 40?µM picloram and 40?mg?l(-1) gum Arabic, in which 64% of the shoot apex explants and 68.8% of the leaf explants yielded somatic embryos. The embryogenic response of the hybrid was higher than that of the E. globulus, especially when NAA was used. The cultures initiated on picloram-containing medium consisted of nodular embryogenic structures surrounded by a mucilaginous coating layer that emerged from a watery callus developed from the initial explants. Cotyledonary somatic embryos were differentiated after subculture of these nodular embryogenic structures on a medium lacking plant growth regulators. Histological analysis confirmed the bipolar organization of the somatic embryos, with shoot and root meristems and closed procambial tissue that bifurcated into small cotyledons. The root pole was more differentiated than the shoot pole, which appeared to be formed by a few meristematic layers. Maintenance of the embryogenic lines by secondary SE was attained by subculturing individual cotyledonary embryos or small clusters of globular and torpedo embryos on medium with 16.11?µM NAA at 4- to 5-week intervals. Somatic embryos converted into plantlets after being transferred to liquid germination medium although plant regeneration remained poor. PMID:25877768

Traditionally, nuclear reprogramming of cells has been performed by transferring somatic cell nuclei into oocytes, by combining\\u000a somatic and pluripotent cells together through cell fusion and through genetic integration of factors through somatic cell\\u000a chromatin. All of these techniques changes gene expression which further leads to a change in cell fate. Here we discuss recent\\u000a advances in generating induced pluripotent

Cotyledon explants from zygotic embryos of Panax ginseng produced somatic embryos on Murashige and Skoog basal medium without growth regulators. Somatic embryos developed directly\\u000a from epidermal cells at the cotyledon base. Somatic embryos were always formed from the side of the cotyledon opposite to\\u000a the one attached to the medium surface regardless of cotyledon orientation. The frequency of somatic embryo

Somaticembryogenesis (SE), which resembles zygotic embryogenesis, is an essential component of the process of plant cell differentiation and embryo development. Although microRNAs (miRNAs) are important regulators of many plant develop- mental processes, their roles in SE have not been thoroughly investigated. In this study, we used deep-sequencing, computational, and qPCR methods to identify, profile, and describe conserved and novel miRNAs involved in longan (Dimocarpus longan) SE. A total of 643 conserved and 29 novel miRNAs (including star strands) from more than 169 miRNA families were identified in longan embryogenic tissue using Solexa sequencing. By combining computational and degradome sequencing approaches, we were able to predict 2063 targets of 272 miRNAs and verify 862 targets of 181 miRNAs. Target annotation revealed that the putative targets were involved in a broad variety of biological processes, including plant metabolism, signal transduction, and stimulus response. Analysis of stage- and tissue-specific expressions of 20 conserved and 4 novel miRNAs indicated their possible roles in longan SE. These miRNAs were dlo-miR156 family members and dlo-miR166c* associated with early embryonic culture developmental stages; dlo-miR26, dlo-miR160a, and families dlo-miR159, dlo-miR390, and dlo-miR398b related to heart-shaped and torpedo- shaped embryo formation; dlo-miR4a, dlo-miR24, dlo-miR167a, dlo-miR168a*, dlo-miR397a, dlo-miR398b.1, dlo-miR398b.2, dlo-miR808 and dlo-miR5077 involved in cotyledonary embryonic development; and dlo-miR17 and dlo-miR2089*-1 that have regulatory roles during longan SE. In addition, dlo-miR167a, dlo-miR808, and dlo-miR5077 may be required for mature embryo formation. This study is the first reported investigation of longan SE involving large-scale cloning, characterization, and expression profiling of miRNAs and their targets. The reported results contribute to our knowledge of somatic embryo miRNAs and provide insights into miRNA biogenesis and expression in plant somatic embryo development. PMID:23593197

Difficulties to develop an easy and reproducible protocol to get healthy and well formed plants from somatic embryos of papaya (Carica papaya L.) had included low germination, callus production at the base of the embryo radicle and the occurrence of hyperhydric plantlets among others, and by consequence unsuccessful transfer to the field. With the aim of improving a propagation method,

Immature zygotic embryos of sunflower (Helianthus annuus L.) produce somatic embryos when cultured on medium supplemented with a cytokinin as the sole source of exogenous growth regulators. The timing of the induction phase and subsequent morphogenic events have been well characterized in previous work. We address here the question of the role of endogenous indole-3-acetic acid (IAA), since auxins are

In angiosperms, the WOX family of transcription factors has important functions in meristem regulation and in control of the\\u000a partitioning of developing embryos into functional domains. In this study, a putative WOX2 homologous gene was isolated from Picea abies, and its expression pattern during somatic embryo development was followed using real-time quantitative reverse transcription\\u000a polymerase chain reaction (qRT-PCR). We used

Somatic embryo induction from immature zygotic embryos followed by embryo development and maturation has been achieved in holm oak (Quercus ilex L.). Different types of explant have been assayed for the induction of somaticembryogenesis. Only immature zygotic embryos, collected in August, were successfully induced. Best results were obtained in Gamborg et al. (1968) medium supplemented with 10 µM BAP

Routine production of large numbers of transgenic plants is required to fully exploit advances in cassava biotechnology and support development of improved germplasm for deployment to farmers. This article describes an improved, high-efficiency transformation protocol for recalcitrant cassava cultivar TME14 preferred in Africa. Factors that favor production of friable embryogenic calli (FEC) were found to be use of DKW medium, crushing of organized embryogenic structures (OES) through 12 mm sized metal wire mesh, washing of crushed OES tissues and short exposure of tyrosine to somatic embryos; and transformation efficiency was enhanced by use of low Agrobacterium density during co-cultivation, co-centrifugation of FEC with Agrobacterium, germination of paramomycin resistant somatic embryos on medium containing BAP with gradual increase in concentration and variations of the frequency of subculture of cotyledonary-stage embryos on shoot elongation medium. By applying the optimized parameters, FEC were produced for cassava cultivar TME14 and transformed using Agrobacterium strain LBA4404 harboring the binary vector pCAMBIA2301. About 7080 independent transgenic lines per ml settled cell volume (SCV) of FEC were regenerated on selective medium. Histochemical GUS assays confirmed the expression of gusA gene in transformed calli, somatic embryos and transgenic plants. The presence and integration of the gusA gene were confirmed by PCR and Southern blot analysis, respectively. RT-PCR analysis of transgenic plants confirmed the expression of gusA gene. This protocol demonstrates significantly enhanced transformation efficiency over existing cassava transformation protocols and could become a powerful tool for functional genomics and transferring new traits into cassava. PMID:26113851

PAPAVER SOMNIFERUM L. tissue cultures, issued from various explants (cotyledons, hypocotyls, roots) derived from plantlets belonging to two genotypes, were established on LS solid medium containing growth regulators (NAA, Kin) in various combinations. Hypocotyls and roots were found to be interesting explants to obtain cellular development. Many roots developed on calli growing on a medium containing NAA (1 mg/l) + Kin (0.1 mg/l) for the PS genotype while somatic proembryos redifferentiated on calli issued from PS 1639 genotype. The same growth substance combination was the most favourable for the production of morphinan alkaloids and papaverine: up to 10 x 10 (-3)% DW in roots redifferentiated from PS calluses. PMID:17260250

Isolated microspores are reprogrammed in vitro by stress, becoming totipotent cells and producing embryos and plants via a process known as microspore embryogenesis. Despite the abundance of data on auxin involvement in plant development and embryogenesis, no data are available regarding the dynamics of auxin concentration, cellular localization and the expression of biosynthesis genes during microspore embryogenesis. This work involved the analysis of auxin concentration and cellular accumulation; expression of TAA1 and NIT2 encoding enzymes of two auxin biosynthetic pathways; expression of the PIN1-like efflux carrier; and the effects of inhibition of auxin transport and action by N-1-naphthylphthalamic acid (NPA) and ?-(p-chlorophenoxy) isobutyric acid (PCIB) during Brassica napus microspore embryogenesis. The results indicated de novo auxin synthesis after stress-induced microspore reprogramming and embryogenesis initiation, accompanying the first cell divisions. The progressive increase of auxin concentration during progression of embryogenesis correlated with the expression patterns of TAA1 and NIT2 genes of auxin biosynthetic pathways. Auxin was evenly distributed in early embryos, whereas in heart/torpedo embryos auxin was accumulated in apical and basal embryo regions. Auxin efflux carrier PIN1-like gene expression was induced in early multicellular embryos and increased at the globular/torpedo embryo stages. Inhibition of polar auxin transport (PAT) and action, by NPA and PCIB, impaired embryo development, indicating that PAT and auxin action are required for microspore embryo progression. NPA also modified auxin embryo accumulation patterns. These findings indicate that endogenous auxin biosynthesis, action and polar transport are required in stress-induced microspore reprogramming, embryogenesis initiation and progression. PMID:25907568

Background Among the many commercial opportunities afforded by somaticembryogenesis (SE), it is the ability to clonally propagate individual plants with rare or elite traits that has some of the most significant implications. This is particularly true for many long-lived species, such as conifers, but whose long generation times pose substantive challenges, including increased recalcitrance for SE as plants age. Identification of a clonal line of somatic embryo-derived trees whose shoot primordia have remained responsive to SE induction for over a decade, provided a unique opportunity to examine the molecular aspects underpinning SE within shoot tissues of adult white spruce trees. Results Microarray analysis was used to conduct transcriptome-wide expression profiling of shoot explants taken from this responsive genotype following one week of SE induction, which when compared with that of a nonresponsive genotype, led to the identification of four of the most differentially expressed genes within each genotype. Using absolute qPCR to expand the analysis to three weeks of induction revealed that differential expression of all eight candidate genes was maintained to the end of the induction treatment, albeit to differing degrees. Most striking was that both the magnitude and duration of candidate gene expression within the nonresponsive genotype was indicative of an intense physiological response. Examining their putative identities further revealed that all four encoded for proteins with similarity to angiosperm proteins known to play prominent roles in biotic defense, and that their high-level induction over an extended period is consistent with activation of a biotic defense response. In contrast, the more temperate response within the responsive genotype, including induction of a conifer-specific dehydrin, is more consistent with elicitation of an adaptive stress response. Conclusions While additional evidence is required to definitively establish an association between SE responsiveness and a specific physiological response, these results suggest that biotic defense activation may be antagonistic, likely related to the massive transcriptional and metabolic reprogramming that it elicits. A major issue for future work will be to determine how and if suppressing biotic defense activation could be used to promote a physiological state more conducive to SE induction. PMID:23937238

Outer hair cells, isolated from mammalian cochleas, are known to respond to electrical stimulation with elongation or contraction of the cell's cylindrical soma. It is assumed that such shape changes, when driven by the cell's receptor potential in vivo, are a part of the feedback process that underlies cochlear amplification. To date it has not been possible to demonstrate somatic shape changes upon normal mechanical stimulation of the cell--i.e., the deflection of its hair bundle. We show here that mechanically induced hair-bundle deflection produces somatic motility of the cell. Such motility is dependent upon a functioning forward transducer process and disappears upon interference with transduction. The motile response also reflects the hair bundle's known directional sensitivity. This demonstration of mechanically driven motility indicates that the cell may possess capabilities to affect its mechanical environment under control of its own receptor potential and, thereby, participate in a local cochlear feedback process. Images Fig. 1 PMID:8378305

Summary Embryogenic callus was induced on segments of young leaves of sugarcane (Saccharum officinarum L.) cultured on Murashige and Skoog's medium supplemented with 0.53.0 mg\\/2,4-D, 5% coconut milk and 38% sucrose. The fourth and fifth leaves, especially their midrib and sheath regions within 5 cm from the leaf base, were most suitable for the induction of embryogenic callus. Many embryoids

Somatic embryos of sunflower (Helianthus annuus) were obtained by placing immature zygotic embryos on a high sucrose (12%) containing medium. The somatic embryos were first observed 6 days after culture and a callus intermediate was not formed. Histological examination revealed the classical stages of embryo development. The somatic embryos proliferated directly from the surface of the zygotic embryos and germinated

\\u000a The cycads (Fig. 1) constitute remnant species of an ancient class of gymnosperms, the cycadophytes, that evolved from the\\u000a free-sporing progymnosperms, which also gave rise to the coniferophytes. According to Gifford & Foster (1989), the cycadophytes\\u000a have included 3 orders of plants, the extinct Cycadeoidales and Pteridospermales (seed ferns), that are known only from the\\u000a fossil record, and the Cycadales,

Embryo formation from callus of Theobroma cacao L. was associated with the changes in relationship between nuclear, nucleolar and cell sizes and the content of basic proteins (FG-FCF-stained). Together with the increase in nuclear size of callus and proembryo cells the increase in the amount of nuclear basic proteins was found. In the callus cells the increase in nucleolar protein content exceeded that in nucleolus size, which led to the rise in basic protein concentration in the nucleolus. However, in the early stage of embryogenesis the increase in protein content was not so marked as that in callus, which indicated that embryogenesis involved a decrease in concentration of nucleolar basic proteins. Differences between callus and proembryo cells were also observed in the concentration of cytoplasmic proteins. The increase in size of callus cells was the same as the increasing amount of cytoplasmic proteins. In proembryos a significant increase in cell size was accompanied by only slight changes in cytoplasmic proteins. The stimulation of embryogenesis by 2,4-D resulted in an increase of nuclear concentration of basic proteins in proembryos. The intensification of embryogenesis involved the decrease of the concentration of nucleolar proteins together with the increase in concentration of basic cytoplasmic proteins. PMID:2464508

Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores. PMID:22197894

For reasons that are unclear the production of embryonic stem cells from ungulates has proved elusive. Here, we describe induced pluripotent stem cells (iPSC) derived from porcine fetal fibroblasts by lentiviral transduction of 4 human (h) genes, hOCT4, hSOX2, hKLF4, and hc-MYC, the combination commonly used to create iPSC in mouse and human. Cells were cultured on irradiated mouse embryonic fibroblasts (MEF) and in medium supplemented with knockout serum replacement and FGF2. Compact colonies of alkaline phosphatase-positive cells emerged after approximately 22 days, providing an overall reprogramming efficiency of approximately 0.1%. The cells expressed porcine OCT4, NANOG, and SOX2 and had high telomerase activity, but also continued to express the 4 human transgenes. Unlike human ESC, the porcine iPSC (piPSC) were positive for SSEA-1, but negative for SSEA-3 and -4. Transcriptional profiling on Affymetrix (porcine) microarrays and real time RT-PCR supported the conclusion that reprogramming to pluripotency was complete. One cell line, ID6, had a normal karyotype, a cell doubling time of approximately 17 h, and has been maintained through >220 doublings. The ID6 line formed embryoid bodies, expressing genes representing all 3 germ layers when cultured under differentiating conditions, and teratomas containing tissues of ectoderm, mesoderm, and endoderm origin in nude mice. We conclude that porcine somatic cells can be reprogrammed to form piPSC. Such cell lines derived from individual animals could provide a means for testing the safety and efficacy of stem cell-derived tissue grafts when returned to the same pigs at a later age. PMID:19541600

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS)

Antibody diversification by somatic hypermutation, gene conversion, and class switch recombination is completely dependent on activation-induced cytidine deaminase (AID). A recent report showing induction of DNA mutations in Escherichia coli by overexpression of AID, Apobec-1, and related members of the RNA-editing cytidine deaminase family suggested that they may directly modify deoxycytidine in DNA in mammalian cells (DNA-editing model). We therefore examined whether Apobec-1 bona fide RNA-editing enzyme could show somatic hypermutation and class switching activities in murine B lymphocytes and fibroblasts. Unlike AID, Apobec-1 was unable to inducesomatic hypermutation or class switching. The results force a reevaluation of the physiological significance of the DNA deaminase activities of AID and Apobec-1 in E. coli and in vitro. PMID:14559972

Almost 9 percent of cosmic rays which strike the earth's atmosphere are alpha particles. As one of the ionizing radiations (IR), its biological effects have been widely studied. However, the plant genomic instability induced by alpha-particle radiation was not largely known. In this research, the Arabidopsis thaliana transgenic for GUS recombination substrate was used to evaluate the genomic instability induced by alpha-particle radiation (3.3MeV). The pronounced effects of systemic exposure to alpha-particle radiation on the somatic homologous recombination frequency (HRF) were found at different doses. The 10Gy dose of radiation induced the maximal HRF which was 1.9-fold higher than the control. The local radiation of alpha-particle (10Gy) on root also resulted in a 2.5-fold increase of somatic HRF in non-radiated aerial plant, indicating that the signal(s) of genomic instability was transferred to non-radiated parts and initiated their genomic instability. Concurrent treatment of seedlings of Arabidopsis thaliana with alpha-particle and DMSO(ROS scavenger) both in systemic and local radiation signifi- cantly suppressed the somatic HR, indicating that the free radicals produced by alpha-particle radiation took part in the production of signal of genomic instability rather than the signal transfer. Key words: alpha-particle radiation, somatic homologous recombination, genomic instability

In plants, programmed cell death (PCD) is an important mechanism that controls normal growth and development as well as many defence responses. At present, research on PCD in different plant species is actively carried out due to the possibilities offered by modern methods in molecular biology and the increasing amount of genome data. The pine seed provides a favourable model for PCD because it represents an interesting inheritance of seed tissues as well as an anatomically well-described embryogenesis during which several tissues die via morphologically different PCD processes. PMID:19826239

Programmed cell death (PCD) in multicellular organisms is a vital process in growth, development, and stress responses that contributes to the formation of tissues and organs. Although numerous studies have defined the molecular participants in apoptotic and PCD cascades, successful identification of early master regulators that target specific cells to live or die is limited. Using Zea mays somaticembryogenesis as a model system, we report that the expressions of two plant hemoglobin (Hb) genes (ZmHb1 and ZmHb2) regulate the cell survival/death decision that influences somaticembryogenesis through their cell-specific localization patterns. Suppression of either of the two ZmHbs is sufficient to induce PCD through a pathway initiated by elevated NO and Zn2+ levels and mediated by production of reactive oxygen species. The effect of the death program on the fate of the developing embryos is dependent on the localization patterns of the two ZmHbs. During somaticembryogenesis, ZmHb2 transcripts are restricted to a few cells anchoring the embryos to the subtending embryogenic tissue, whereas ZmHb1 transcripts extend to several embryonic domains. Suppression of ZmHb2 induces PCD in the anchoring cells, allowing the embryos to develop further, whereas suppression of ZmHb1 results in massive PCD, leading to abortion. We conclude that regulation of the expression of these ZmHbs has the capability to determine the developmental fate of the embryogenic tissue during somaticembryogenesis through their effect on PCD. This unique regulation might have implications for development and differentiation in other species. PMID:24784758

Inducingsomatic embryogensis from jojoba [Simmondsia chinensis (Link) Schneider] explants to produce artificial seeds in the laboratory (in vitro) may prove highly profitable, as the seeds\\u000a contain a characteristic liquid wax of economic importance in industry, nutrition and medicine. Thus, there is a need to examine\\u000a the effect of the factors involved in the in vitro process on the quality

Somatic sensations induced by placebos are a frequent phenomenon whose etiology and clinical relevance remains unknown. In this study, we have evaluated the quantitative, qualitative, spatial, and temporal characteristics of placebo-inducedsomatic sensations in response to three different placebo interventions: (1) placebo irritant solution, (2) placebo laser stimulation, and (3) imagined laser stimulation. The quality and intensity of evoked sensations were assessed using the McGill pain questionnaire and visual analogue scales (VAS), while subjects' sensation drawings processed by a geographic information system (GIS) were used to measure their spatial characteristics. We found that all three interventions are capable of producing robust sensations most frequently described as "tingling" and "warm" that can reach consider-able spatial extent (? 205mm²) and intensity (? 80/100 VAS). Sensations from placebo stimulation were often referred to areas remote from the stimulation site and exhibit considerable similarity with referred pain. Interestingly, there was considerable similarity of qualitative features as well as spatial patterns across subjects and placebos. However, placebo laser stimulation elicited significantly stronger and more widespread sensations than placebo irritant solution. Finally, novelty seeking, a character trait assessed by the Temperament and Character Inventory and associated with basal dopaminergic activity, was less pronounced in subjects susceptible to report placebo-induced sensations. Our study has shown that placebo-induced sensations are frequent and can reach considerable intensity and extent. As multiple somatosensory subsystems are involved despite the lack of peripheral stimulus, we propose a central etiology for this phenomenon. PMID:25901350

Protoplasts of navel orange, isolated from embryogenic nucellar cell suspension culture, were fused with protoplasts of grapefruit isolated from leaf tissue. The fusion products were cultured in the hormone-free medium containing 0.6 M sucrose. Under the culture conditions, somaticembryogenesis of navel orange protoplasts was suppressed, while cell division of grapefruit mesophyll protoplasts was not induced. Six embryoids were obtained and three lines regenerated to complete plants through embryogenesis. Two of the regenerated lines exhibited intermediate morphological characteristics of the parents in the leaf shape. Chromosome counts showed that these regenerated plants had expected 36 chromosomes (2n=2x=18 for each parent). The rDNA analysis using biotin-labeled rRNA probes confirmed the presence of genomes from both parents in these plants. This somatic hybridization system would be useful for the practical Citrus breeding. PMID:24225818

Summary Calli were induced from leaf expiants of aDactylis glomerata L. (orchardgrass) genotype which has a high capacity for somaticembryogenesis. After 7 months culture on SH medium containing NaCl, a line was selected which was tolerant to 200 mM NaCl. When both selected and nonselected calli were maintained for 56 days on media containing 0 to 300 mM NaCl,

Microspore embryogenesis involves reprogramming of the pollen immature cell towards embryogenesis. We have identified and characterized a collection of 14 genes induced along different morphological phases of microspore-derived embryo development in wheat (Triticum aestivum L.) anther culture. SERKs and FLAs genes previously associated with somaticembryogenesis and reproductive tissues, respectively, were also included in this analysis. Genes involved in signalling mechanisms such as TaTPD1-like and TAA1b, and two glutathione S-transferase (GSTF2 and GSTA2) were induced when microspores had acquired a 'star-like' morphology or had undergone the first divisions. Genes associated with control of plant development and stress response (TaNF-YA, TaAGL14, TaFLA26, CHI3, XIP-R; Tad1 and WALI6) were activated before exine rupture. When the multicellular structures have been released from the exine, TaEXPB4, TaAGP31-like and an unknown embryo-specific gene TaME1 were induced. Comparison of gene expression, between two wheat cultivars with different response to anther culture, showed that the profile of genes activated before exine rupture was shifted to earlier stages in the low responding cultivar. This collection of genes constitutes a value resource for study mechanism of intra-embryo communication, early pattern formation, cell wall modification and embryo differentiation. PMID:23839308

Functional analysis of multiple genes is key to understanding gene regulatory networks controlling embryonic development. We have developed an integrated vector system for inducible gene silencing by shRNAmir-mediated RNA interference in mouse embryos, as a fast method for dissecting mammalian gene function. For validation of the vector system, we generated mutant phenotypes for Brachyury, Foxa2 and Noto, transcription factors which play pivotal roles in embryonic development. Using a series of Brachyury shRNAmir vectors of various strengths we generated hypomorphic and loss of function phenotypes allowing the identification of Brachyury target genes involved in trunk development. We also demonstrate temporal control of gene silencing, thus bypassing early embryonic lethality. Importantly, off-target effects of shRNAmir expression were not detectable. Taken together, the system allows the dissection of gene function at unprecedented detail and speed, and provides tight control of the genetic background minimizing intrinsic variation. PMID:20350929

Parasitic helminths polarize immune response of their vertebrate hosts towards anti-inflammatory Th2 type and therefore it is hypothesized that they may suppress the inflammatory conditions in autoimmune disorders. The present study was undertaken to investigate in vivo immunomodulatory and therapeutic potential of somatic antigens (Ag) of liver infecting digenetic trematodes [Fasciola gigantica (Fg) and Gigantocotyle explanatum (Ge)] in collagen-induced arthritic (CIA) Wistar rats. The CIA rats were administered subcutaneously with different doses (50 ?g, 100 ?g and 150 ?g) of somatic antigens of Fg and Ge, daily for 21 days, the time period required to establish infection in natural host (Bubalus bubalis). Thereafter, the control, diseased and treated rats were compared for different parameters viz. hind paw thickness; serum interleukins, IL-4 and IL-10, tumor necrosis factor-? (TNF-?) and interferon-? (IFN-?); expression level of matrix metalloproteinases (MMPs) -2, -9, -13 and nitric oxide (NO) in knee joints and patellar morphology. The CIA rats treated with different antigens, Fg-Ag and Ge-Ag, show significant amelioration of the disease by down regulation of serum TNF-? and IFN-? (p< 0.05) and upregulation of IL-4 and IL-10 cytokines (p< 0.05); inhibition (p< 0.05) of MMPs (-2,-9,-13) and NO in knee joints and improved patellar morphology with decreased synovial hypertrophy and reduced infiltration of ploymorphonuclear cells. The activity of pro as well as active MMPs (-2 and -9) and active MMP-13 in knee joints of CIA rats was very high compared to the control and treatment groups, suggesting the extent of collagen degradation in CIA rats. Interestingly, the highest dose (150 ?g) of Ge-Ag almost wiped out MMP-13 expression. The overall findings suggest that the somatic proteins of Ge-Ag appeared to be therapeutically more effective than Fg-Ag, reflecting interspecific molecular differences which could contribute to the ability of these worms to successfully ameliorate the pathology of CIA. PMID:25992888

Parasitic helminths polarize immune response of their vertebrate hosts towards anti-inflammatory Th2 type and therefore it is hypothesized that they may suppress the inflammatory conditions in autoimmune disorders. The present study was undertaken to investigate in vivo immunomodulatory and therapeutic potential of somatic antigens (Ag) of liver infecting digenetic trematodes [Fasciola gigantica (Fg) and Gigantocotyle explanatum (Ge)] in collagen-induced arthritic (CIA) Wistar rats. The CIA rats were administered subcutaneously with different doses (50 ?g, 100 ?g and 150 ?g) of somatic antigens of Fg and Ge, daily for 21 days, the time period required to establish infection in natural host (Bubalus bubalis). Thereafter, the control, diseased and treated rats were compared for different parameters viz. hind paw thickness; serum interleukins, IL-4 and IL-10, tumor necrosis factor-? (TNF-?) and interferon-? (IFN-?); expression level of matrix metalloproteinases (MMPs) -2, -9, -13 and nitric oxide (NO) in knee joints and patellar morphology. The CIA rats treated with different antigens, Fg-Ag and Ge-Ag, show significant amelioration of the disease by down regulation of serum TNF-? and IFN-? (p< 0.05) and upregulation of IL-4 and IL-10 cytokines (p< 0.05); inhibition (p< 0.05) of MMPs (-2,-9,-13) and NO in knee joints and improved patellar morphology with decreased synovial hypertrophy and reduced infiltration of ploymorphonuclear cells. The activity of pro as well as active MMPs (-2 and -9) and active MMP-13 in knee joints of CIA rats was very high compared to the control and treatment groups, suggesting the extent of collagen degradation in CIA rats. Interestingly, the highest dose (150 ?g) of Ge-Ag almost wiped out MMP-13 expression. The overall findings suggest that the somatic proteins of Ge-Ag appeared to be therapeutically more effective than Fg-Ag, reflecting interspecific molecular differences which could contribute to the ability of these worms to successfully ameliorate the pathology of CIA. PMID:25992888

Channelrhodopsins are light-gated ion channels involved in the photoresponses of microalgae. Here, we describe the characterization of two channelrhodopsins, Volvox channelrhodopsin-1 (VChR1) and VChR2, from the multicellular green alga Volvox carteri. Both are encoded by nuclear single copy genes and are highly expressed in the small biflagellated somatic cells but not in the asexual reproductive cells (gonidia). Expression of both VChRs increases after cell cleavage and peaks after completion of embryogenesis, when the biosynthesis of the extracellular matrix begins. Likewise, expression of both transcripts increases after addition of the sex-inducer protein, but VChR2 is induced much more than VChR1. The expression of VChR1 is specifically promoted by extended dark periods, and heat stress reduces predominantly VChR1 expression. Expression of both VChRs increased under low light conditions, whereas cold stress and wounding reduced expression. Both VChRs were spectroscopically studied in their purified recombinant forms. VChR2 is similar to the ChR2 counterpart from Chlamydomonas reinhardtii with respect to its absorption maximum (460 nm) and photocycle dynamics. In contrast, VChR1 absorbs maximally at 540 nm at low pH (D540), shifting to 500 nm at high pH (D500). Flash photolysis experiments showed that after light excitation, the D540 dark state bleaches and at least two photoproducts, P600 and P500, are sequentially populated during the photocycle. We hypothesize that VChR2 is a general photoreceptor that is responsible for the avoidance of blue light and might play a key role in sexual development, whereas VChR1 is the main phototaxis photoreceptor under vegetative conditions, as it is more specifically adapted to environmental conditions and the developmental stages of Volvox. PMID:19641026

The production of pluripotent stem cells (iPSCs) for therapeutic applications will require practical methods to achieve tight temporal and quantitative control of reprogramming factor (RF) expression, while avoiding the mutagenic potential of gene transfer. Toward this end, we have developed cell-permeable RF proteins (CP-RFs) incorporating newly developed macromolecule transduction domains (MTDs). Treatment of human dermal fibroblasts (HDFs) with combinations of cell-permeable OCT4, SOX2, KLF4, CMYC and either NANOG or LIN28 proteins induced the outgrowth of stem cell-like colonies (iSCs). iSC colonies generated with CP-RFs resembled embryonic stem cells with regard to morphology, biomarker expression, and extended capacity for self-renewal, but failed to expand as iPSC or ES cell lines. Partial reprogramming appears to be a common response to protein-based delivery of programming factors into somatic cells. PMID:24618595

This study presents the analysis of chemically-inducedsomatic mutations and chromosomal damage in the eye imaginal discs of Drosophila larvae, assayed later as twin (TS) and single light (LS) mosaic spots in the adult eyes. Regarding the question as to what kind of DNA alterations contribute to somatic cell mutagenicity, the approach followed here has been to investigate the possible differences in response between male (hemizygous for an X) and female (homozygous) larvae, rod-X/rod-X versus ring-X/rod-X genotypes and inversion-heterozygotes versus genotypes not carrying an inversion. The systems chosen for this analysis were the white-coral/white (wco/w) and the white+/white (w+/w) eye mosaic system. The principle findings with 12 mutagens of different modes of action are as follows: (1) At least 98% of all TS and LS induced by cisplatin (DDP) in wco/w female larvae and about 95% of those by formaldehyde (FA) appear as the result of recombinogenic activity between the two homologous X-chromosomes. The corresponding estimates for MMS, EMS and ENU are 81%, 73% and 61%, respectively. (2) The long scS1L sc8R inversion, which also contains In(1)dl-49, suppresses induction of TS to 83-93%. There was also a sharp decline in the frequency of LS in inversion heterozygotes for DDP (91%), FA (86%), MMS (52%) and EMS (47%). (3) Ethylnitrosourea (ENU) was the mutagen for which introduction of the inverted chromosome reduced only slightly (23%) the frequency of LS, indicating that the majority of them were somatic mutations (and deletions) at the white locus. (4) In w/RX females heterozygous for a ring-X chromosome, the frequency of LS was only approximately one tenth of that of the control (w+/w) group, after exposure to MMS or DDP. The explanation is that exchange processes involving the ring frequently lead to genetic imbalance with subsequent cell killing. PMID:3116423

There was a linear relation between an increase in DNA content and size of nuclei, nucleoli and cells in callus and proembryos (Theobroma cacao L.). In callus the increase of DNA content was accompanied by proportional increase in nuclear size whereas in proembryos the increase in nuclear size did not match the increasing amount of DNA. The stimulation of embryogenesis by 10(-2) mg/l 2,4-D was associated with increase in nuclear and nucleolar size and with decrease in cell sizes. Inhibition of embryogenesis by 1.0 mg/l 2,4-D+10% coconut water did not change nuclear size, but increased cell size in relation to the control. The process of embryo formation was accompanied by changes in relationship between nuclear, nucleolar and cell size and the total (DNFB-stained) proteins content. In callus as well as in proembryo the increase in total protein content in nucleus was not equivalent to the increasing sizes of nuclei which leads to the decrease in nuclear protein concentration. Similar situation was observed for nucleoli. Differences were found in the concentration of cytoplasmic proteins between the callus and proembryo cells. The stimulation of embryogenesis by low concentration of 2,4-D resulted in decrease in concentration of total proteins in nuclei and nucleoli and the increase in cytoplasm. PMID:3220146

Background Although recent studies have identified genes expressed in human embryonic stem cells (hESCs) that induce pluripotency, the molecular underpinnings of normal stem cell function remain poorly understood. The high mobility group A1 (HMGA1) gene is highly expressed in hESCs and poorly differentiated, stem-like cancers; however, its role in these settings has been unclear. Methods/Principal Findings We show that HMGA1 is highly expressed in fully reprogrammed iPSCs and hESCs, with intermediate levels in ECCs and low levels in fibroblasts. When hESCs are induced to differentiate, HMGA1 decreases and parallels that of other pluripotency factors. Conversely, forced expression of HMGA1 blocks differentiation of hESCs. We also discovered that HMGA1 enhances cellular reprogramming of somatic cells to iPSCs together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC  OSKM). HMGA1 increases the number and size of iPSC colonies compared to OSKM controls. Surprisingly, there was normal differentiation in vitro and benign teratoma formation in vivo of the HMGA1-derived iPSCs. During the reprogramming process, HMGA1 induces the expression of pluripotency genes, including SOX2, LIN28, and cMYC, while knockdown of HMGA1 in hESCs results in the repression of these genes. Chromatin immunoprecipitation shows that HMGA1 binds to the promoters of these pluripotency genes in vivo. In addition, interfering with HMGA1 function using a short hairpin RNA or a dominant-negative construct blocks cellular reprogramming to a pluripotent state. Conclusions Our findings demonstrate for the first time that HMGA1 enhances cellular reprogramming from a somatic cell to a fully pluripotent stem cell. These findings identify a novel role for HMGA1 as a key regulator of the stem cell state by inducing transcriptional networks that drive pluripotency. Although further studies are needed, these HMGA1 pathways could be exploited in regenerative medicine or as novel therapeutic targets for poorly differentiated, stem-like cancers. PMID:23166588

Mesophyl protoplasts of two genotypes of cultivated tomato (Lycopersicon esculentum Mill.) and one of its wild relative species (Lycopersicon peruvianum Mill.) were fused by using electrofusion and polyethyleneglycol-induced fusion. Forty-three fertile tetraploid somatic hybrid plants, each deriving from separate calli, were recovered from both fusion procedures. Electrofusion appeared more efficient than chemical fusion for the production of somatic hybrids. These

Imprinting disorders induced by somatic cell nuclear transfer (SCNT) usually lead to the abnormalities of cloned animals and low cloning efficiency. Histone deacetylase inhibitors have been shown to improve gene expression, genomic methylation reprogramming and the development of cloned embryos, however, the imprinting statuses in these treated embryos and during their subsequent development remain poorly studied. In this study, we investigated the dynamics of H19/Igf2 methylation and transcription in porcine cloned embryos treated with trichostatin A (TSA), and examined H19/Igf2 imprinting patterns in cloned fetuses and piglets. Our results showed that compared with the maintenance of H19/Igf2 methylation in fertilized embryos, cloned embryos displayed aberrant H19/Igf2 methylation and lower H19/Igf2 transcripts. When TSA enhanced the development of cloned embryos, the disrupted H19/Igf2 imprinting was largely rescued in these treated embryos, more similar to those detected in fertilized counterparts. Further studies displayed that TSA effectively rescued the disrupted imprinting of H19/Igf2 in cloned fetuses and piglets, prevented the occurrence of cloned fetus and piglet abnormalities, and enhanced the full-term development of cloned embryos. In conclusion, our results demonstrated that aberrant imprinting induced by SCNT led to the abnormalities of cloned fetuses and piglets and low cloning efficiency, and TSA rescued the disrupted imprinting in cloned embryos, fetuses and piglets, and prevented the occurrence of cloned fetus and piglet abnormalities, thereby improving the development of cloned embryos. This study would have important implications in improving cloning efficiency and the health of cloned animals. PMID:25962071

Somatic hypermutation (SHM) occurs at a specific B-cell differentiation stage, during the germinal centre reaction, and provides a means to diversify and shape the antibody repertoire of the adaptive immune system. Burkitt-Lymphoma (BL) is a germinal centre derived B-cell malignancy. Presumably deregulation of the somatic hypermutation- and\\/or class switch recombination process causes a translocation between the myc-locus and one of

Induced pluripotent stem cells (iPSCs) derived from somatic cells (SCs) and embryonic stem cells (ESCs) provide promising resources for regenerative medicine and medical research, leading to a daily identification of new cell lines. However, an efficient system to discriminate the different types of cell lines is lacking. Here, we develop a quantitative system to discriminate the three cell types, iPSCs, ESCs, and SCs. The system consists of DNA-methylation biomarkers and mathematical models, including an artificial neural network and support vector machines. All biomarkers were unbiasedly selected by calculating an eigengene score derived from analysis of genome-wide DNA methylations. With 30 biomarkers, or even with as few as 3 top biomarkers, this system can discriminate SCs from pluripotent cells (PCs, including ESCs and iPSCs) with almost 100% accuracy. With approximately 100 biomarkers, the system can distinguish ESCs from iPSCs with an accuracy of 95%. This robust system performs precisely with raw data without normalization as well as with converted data in which the continuous methylation levels are accounted. Strikingly, this system can even accurately predict new samples generated from different microarray platforms and the next-generation sequencing. The subtypes of cells, such as female and male iPSCs and fetal and adult SCs, can also be discriminated with this method. Thus, this novel quantitative system works as an accurate framework for discriminating the three cell types, iPSCs, ESCs, and SCs. This strategy also supports the notion that DNA-methylation generally varies among the three cell types. PMID:23418520

Theobroma cacao L., an economically important crop for developing countries, can be experimentally propagated by somaticembryogenesis. Because\\u000a of their potential roles in embryogenesis, a gene candidate strategy was initiated to find gene homologues of the members\\u000a of the leafy cotyledon family of transcription factors. A homologue of the leafy cotyledon1-like gene, that encodes the HAP 3 subunit of the

We have shown that two 27-kD proteins, designated as WAP27A and WAP27B, were abundantly accumulated in endoplasmic reticulum-enriched fractions isolated from cortical parenchyma cells of mulberry tree (Morus bombycis Koidz.) during winter (N. Ukaji, C. Kuwabara, D. Takezawa, K. Arakawa, S. Yoshida, S. Fujikawa [1999] Plant Physiol 120: 480489). In the present study, cDNA clones encoding WAP27A and WAP27B were isolated and characterized. The deduced amino acid sequences of WAP27A and WAP27B cDNAs had 12 repeats of an 11-mer amino acid motif that was the common feature of group 3 late-embryogenesis-abundant proteins. Under field conditions, transcripts of WAP27 genes were initially detected in mid-October, reached maximum level from mid-November to mid-December, and then gradually decreased. The transcript levels of WAP27 genes in cortical parenchyma cells harvested in October was drastically induced by cold treatment within a few days, whereas those in cortical parenchyma cells harvested in August were low even by cold treatment for 3 weeks. Immunocytochemical analysis by electron microscopy confirmed that WAP27 was localized specifically in vesicular-form ER and also localized in dehydration-induced multiplex lamellae-form ER. The role of WAP27 in the ER is discussed in relation to acquisition of freezing tolerance of cortical parenchyma cells in mulberry tree during winter. PMID:11500557

Summary Somatic embryos produced in vitro may exhibit structural abnormalities that affect their subsequent germination and conversion into plants. To assess the influence of auxin type on embryo initiation and development, a morphological and histological comparison was made of pecan (Carya illinoinensis) somatic embryogenic cultures induced on media with naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (2,4-D), using light and scanning electron

The development of a complete organism from a single cell involves extraordinarily complex orchestration of biological processes that vary intricately across space and time. Systems biology seeks to describe how all elements of a biological system interact in order to understand, model, and ultimately predict aspects of emergent biological processes. Embryogenesis represents an extraordinary opportunity  and challenge  for the application of systems biology. Systems approaches have already been used successfully to study various aspects of development, from complex intracellular networks to 4D models of organogenesis. Going forward, great advancements and discoveries can be expected from systems approaches applied to embryogenesis and developmental biology. PMID:20003850

Our previous studies provided evidence that mammalian mitochondrial DNA (mtDNA) mutations that cause mitochondrial respiration defects behave in a recessive manner, because the induction of respiration defects could be prevented with the help of a small proportion (10%-20%) of mtDNA without the mutations. However, subsequent studies found the induction of respiration defects by the accelerated accumulation of a small proportion of mtDNA with various somatic mutations, indicating the presence of mtDNA mutations that behave in a dominant manner. Here, to provide the evidence for the presence of dominant mutations in mtDNA, we used mouse lung carcinoma P29 cells and examined whether some mtDNA molecules possess somatic mutations that dominantly induce respiration defects. Cloning and sequence analysis of 40-48 mtDNA molecules from P29 cells was carried out to screen for somatic mutations in protein-coding genes, because mutations in these genes could dominantly regulate respiration defects by formation of abnormal polypeptides. We found 108 missense mutations existing in one or more of 40-48 mtDNA molecules. Of these missense mutations, a T15091C mutation in the Cytb gene was expected to be pathogenic due to the presence of its orthologous mutation in mtDNA from a patient with cardiomyopathy. After isolation of many subclones from parental P29 cells, we obtained subclones with various proportions of T15091C mtDNA, and showed that the respiration defects were induced in a subclone with only 49% T15091C mtDNA. Because the induction of respiration defects could not be prevented with the help of the remaining 51% mtDNA without the T15091C mutation, the results indicate that the T15091C mutation in mtDNA dominantly induced the respiration defects. PMID:26072375

Non-coding RNAs (ncRNAs) are emerging as key regulators of embryogenesis. They control embryonic gene expression by several means, ranging from microRNA-induced degradation of mRNAs to long ncRNA-mediated modification of chromatin. Many aspects of embryogenesis seem to be controlled by ncRNAs, including the maternalzygotic transition, the maintenance of pluripotency, the patterning of the body axes, the specification and differentiation of cell

Reprogramming of somatic cells into induced pluripotent stem (iPS) cells by defined pluripotency and self-renewal factors has taken stem cell technology to the forefront of regenerative medicine. However, a number of challenges remain in the field including efficient protocols and the threat of cancer. Reprogramming of plant somatic cells to plant embryonic stem cells using a combination of two plant hormones was discovered in 1957 and has been a routine university laboratory practical for over 30 years. The plant hormones responsible for cell reprogramming to pluripotency, indole-3-acetic acid (IAA) and isopentenyl adenosine (IPA), are present in human cells, leading to the exciting possibility that plant hormones might reprogram mammalian cells without genetic factors. We found that plant hormones on their own could not reprogram mammalian cells but increase the efficiency of the early formation of iPS cells combined with three defined genetic factors during the first 3 weeks of reprogramming by accelerating the cell cycle and regulating pluripotency genes. Moreover, the cytokinin IPA, a known human anticancer agent, reduced the threat of cancer of iPS cell in vitro by regulating key cancer and stem cell-related genes, most notably c-Myc and Igf-1. In conclusion, the plant hormones, auxin and cytokinin, are new small chemicals useful for enhancing early reprogramming efficiency of mammalian cells and reducing the threat of cancer from iPS cells. These findings suggest a novel role for plant hormones in the biology of mammalian cell plasticity. PMID:24251409

The distribution of poly(A)-containing RNA (poly(A)+RNA) in pollen grains of Hyoscyamus niger during normal gametophytic development and embryogenic development induced by culture of anther segments was followed by in situ hybridization with (3H)- polyuridylic acid as a probe . No binding of the isotope occurred in pollen grains during the uninucleate phase of their development . Although (3H)polyuridylic acid binding

Somatic embryos from callus of Salix viminalis L. L. Grönroos, S. von Arnold T. Ericsson Department is in preparation and will be pub- lished elsewhere. Somaticembryogenesis in pistil callus of basket willow (Salix of embryogenic cal- lus obtained from Salix in the future, the embryogenic callus and the other callus types

The effect of the auxins dicamba (3,6-dichloro-2-methoxybenzoic acid) and picloram (4-amino-3,5,6-trichloropicolinic acid) on callus growth and embryogenesis in Phoenix dactylifera L. was investigated. Maximum callus fresh weight was obtained in nutrient medium enriched with 200 µm picloram. Somaticembryogenesis and subsequent plant regeneration was achieved following transfer of such calli to hormone-free medium. Germination of the somatic embryos was influenced

Embryogenic cultures were initiated from immature pecan zygotic embryos. Explants were induced for one week on Woody Plant Medium with either ?-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid at 2, 6 or 12 mg/l, then subcultured monthly to fresh basal medium. Observations were made on callus production, embryo formation, and embryo morphology. Somatic embryo morphology and overall callus proliferation were affected by auxin type. Callus proliferation was less extensive and more somatic embryos resembling zygotic embryos were obtained from cultures initiated with ?-naphthaleneacetic acid than with 2,4-dichlorophenoxyacetic acid. Repetitive somaticembryogenesis was obtained in all auxin treatments. Conversion into plantlets was affected by somatic embryo morphology in that embryos with poorly developed apices exhibited lower percentages of conversion than those with well developed single or multiple apices. Consequently, although more embryos were obtained with 2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid was the superior auxin for production of somatic embryos more likely to convert into plants. PMID:24196238

\\u000a Micropropagation technique is used for rapid shoot proliferation of date palm. Somaticembryogenesis is meant for clonal propagation\\u000a of date palm and genetic gains can be captured through it, which is rather difficult by zygotic embryo due to its heterozygous\\u000a nature. Genetic variability is highly desirable for the genetic improvement of crops, which can be either spontaneous or induced\\u000a by

The distribution of poly(A)-containing RNA [poly(A)+RNA] in pollen grains of Hyoscyamus niger during normal gametophytic development and embryogenic development induced by culture of anther segments was followed by in situ hybridization with [3H]-polyuridylic acid as a probe. No binding of the isotope occurred in pollen grains during the uninucleate phase of their development. Although [3H]polyuridylic acid binding sites were present in the generative and vegetative cells of maturing pollen grains, they almost completely disappeared from mature grains ready to germinate. During pollen germination, poly(A)+RNA formation was transient and was due to the activity of the generative nucleus, whereas the vegetative nucleus and the sperm cells failed to interact with the applied probe. In cultured anther segments, moderate amounts of poly(A)+RNA were detected in the uninucleate, nonvacuolate, embryogenically determined pollen grains. Poly(A)+RNA accumulation in these grains was sensitive to actinomycin D, suggesting that it represents newly transcribed mRNA. After the first haploid mitosis in the embryogenically determined pollen grains, only those grains in which the generative nucleus alone or along with the vegetative nucleus accumulated poly(A)+RNA in the surrounding cytoplasm were found to divide in the embryogenic pathway. Overall, the results suggest that, in contrast to normal gametophytic development, embryogenic development in the uninucleate pollen grains of cultured anther segments of H. niger is due to the transcriptional activation of an informational type of RNA. Subsequent divisions in the potentially embryogenic binucleate pollen grains appeared to be mediated by the continued synthesis of mRNA either in the generative nucleus or in both the generative and vegetative nuclei. PMID:6166618

Somatic cell nuclear transfer (SCNT) has generally demonstrated that a differentiated cell can convert into a undifferentiated or pluripotent state. In the SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor nuclei to the recipient cytoplasm of matured oocytes. However, because the efficiency of SCNT still remains low, a combination of SCNT technique with the ex-ovo method may improve the normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from the germinal vesicle (GV) stage Siberian sturgeon oocytes prior to their use as nuclear donor for SCNT would improve in vitro development. A reversible permeability protocol with 4 ?g/mL of digitonin for 2 min at 4°C in order to deliver Siberian sturgeon oocyte extract (SOE) to porcine fetal fibroblasts (PFFs) was carried out. As results, the intensity of H3K9ac staining in PFFs following treatment of SOE for 7 h at 18°C was significantly increased but the intensity of H3K9me3 staining in PFFs was significantly decreased as compared with the control (p<0.05). Additionally, the level of histone acetylation in SCNT embryos at the zygote stage was significantly increased when reconstructed using SOE-treated cells (p<0.05), similar to that of IVF embryos at the zygote stage. The number of apoptotic cells was significantly decreased and pluripotency markers (Nanog, Oct4 and Sox2) were highly expressed in the blastocyst stage of SCNT embryos reconstructed using SOE-treated cells as nuclear donor (p<0.05). And there was observed a better development to the blastocyst stage in the SOE-treated group (p<0.05). Our results suggested that pre-treatment of cells with SOE could improve epigenetic reprogramming and the quality of porcine SCNT embryos. PMID:25049951

Cell transplantation is an attractive potential therapy for heart diseases. For example, myocardial infarction (MI) is a leading cause of mortality in many countries. Numerous medical interventions have been developed to stabilize patients with MI and, although this has increased survival rates, there is currently no clinically approved method to reverse the loss of cardiac muscle cells (cardiomyocytes) that accompanies this disease. Cell transplantation has been proposed as a method to replace cardiomyocytes, but a safe and reliable source of cardiogenic cells is required. An ideal source would be the patients' own somatic tissue cells, which could be converted into cardiogenic cells and transplanted into the site of MI. However, these are difficult to produce in large quantities and standardized protocols to produce cardiac cells would be advantageous for the research community. To achieve these research goals, small molecules represent attractive tools to control cell behavior. In this editorial, we introduce the use of small molecules in stem cell research and summarize their application to the induction of cardiogenesis in non-cardiac cells. Exciting new developments in this field are discussed, which we hope will encourage cardiac stem cell biologists to further consider employing small molecules in their culture protocols. PMID:25810812

Embryogenic callus developed in 55% of the mature embryo explants of Norway spruce (Picea abies L.) growing on a LP medium minus the amino acids and sugars (except sucrose). This is the highest reported yield of embryogenic callus from mature embryos of P. abies that has ever been reported. Callus induction from either the middle or the end of the

This article reports the culture and plant regeneration of Tripsacum dactyloides. Mature embryos of Tripsacum dactyloides dactyloides were used to obtain embryogenic callus cultures. Currently, 180 normal plants have been regenerated from these cultures. Callus was initiated on MS medium supplemented with dicamba (10 µmol or 20 µmol) and sucrose (3% or 6%), and plants were regenerated on hormone free

Nuclear factor Y (NF-Y) is a highly conserved transcription factor presented in all eukaryotic organisms, and is a heterotrimer consisting of three subunits: NF-YA, NF-YB, and NF-YC. In Arabidopsis, these three subunits are encoded by multigene families. The best-studied member of the NF-Y transcription factors is LEAFY COTYLEDON1 (LEC1), a NF-YB family member, which plays a critical role in embryogenesis and seed maturation. However, the function of most NF-Y genes remains elusive. Here, we report the characterization of four genes in the NF-YA family. We found that a gain-of-function mutant of NF-YA1 showed defects in male gametogenesis and embryogenesis. Consistently, overexpression of NF-YA1, 5, 6, and 9 affects male gametogenesis, embryogenesis, seed morphology, and seed germination, with a stronger phenotype when overexpressing NF-YA1 and NF-YA9. Moreover, overexpression of these NF-YA genes also causes hypersensitivity to abscisic acid (ABA) during seed germination, retarded seedling growth, and late flowering at different degrees. Intriguingly, overexpression of NF-YA1, 5, 6, and 9 is sufficient to induce the formation of somatic embryos from the vegetative tissues. However, single or double mutants of these NF-YA genes do not have detectable phenotype. Collectively, these results provide evidence that NF-YA1, 5, 6, and 9 play redundant roles in male gametophyte development, embryogenesis, seed development, and post-germinative growth. PMID:22933713

Metabolism is vital to every aspect of cell function, yet the metabolome of induced pluripotent stem cells (iPSCs) remains largely unexplored. Here we report, using an untargeted metabolomics approach, that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells, and that is characterized by changes in metabolites involved in cellular

Liver cancer in men is the second leading cause of cancer death and hepatocellular carcinoma (HCC) accounts for 70%-85% of the total liver cancer worldwide. Chronic infection with hepatitis B virus (HBV) is the major cause of HCC. Chronic, intermittently active inflammation provides fertile field for mutation, selection, and adaptation of HBV and the infected hepatocytes, a long-term evolutionary process during HBV-induced carcinogenesis. HBV mutations, which are positively selected by insufficient immunity, can promote and predict the occurrence of HCC. Recently, advanced sequencing technologies including whole genome sequencing, exome sequencing, and RNA sequencing provide opportunities to better under-stand the insight of how somatic mutations, structure variations, HBV integrations, and epigenetic modifications contribute to HCC development. Genomic variations of HCC caused by various etiological factors may be different, but the common driver mutations are important to elucidate the HCC evolutionary process. Genome-wide analyses of HBV integrations are helpful in clarifying the targeted genes of HBV in carcinogenesis and disease progression. RNA sequencing can identify key molecules whose expressions are epigenetically modified during HCC evolution. In this review, we summarized the current findings of next generation sequencings for HBV-HCC and proposed a theory framework of Cancer Evolution and Development based on the current knowledge of HBV-induced HCC to characterize and interpret evolutionary mechanisms of HCC and possible other cancers. Understanding the key viral and genomic variations involved in HCC evolution is essential for generating effective diagnostic, prognostic, and predictive biomarkers as well as therapeutic targets for the interventions of HBV-HCC. PMID:25646075

As a product of asexual reproduction in plants, the somatic embryo (SE) differentiates into a new plantlet via a zygotic embryogenesis-like process. Here, we present the phenotypic and cellular differences between SEs and zygotic embryos (ZEs) revealed by histological section scanning using three parallel development stages of the two types of embryos of cotton (Gossypium hirsutum cv. YZ1), including globular, torpedo and cotyledonary-stages. To identify the molecular characteristics of SE development in cotton, the digital gene expression system was used to profile the genes active during SE and ZE development. A total of 4242 differentially expressed genes (DEGs) were identified in at least one developmental stage. Expression pattern and functional classification analysis based on these DEGs reveals that SE development exhibits a transcriptional activation of stress responses. RT-PCR analysis further confirmed enhanced expression levels of stress-related genes in SEs than in ZEs. Experimental stress treatment, induced by NaCl and ABA, accelerated SE development and increased the transcription of genes related to stress response, in parallel with decelerated proliferation of embryogenic calluses under stress treatment. Our data reveal that SE development involves the activation of stress responses, which we suggest may regulate the balance between cell proliferation and differentiation. These results provide new insight into the molecular mechanisms of SE development and suggest strategies that can be used for regulating the developmental processes of somaticembryogenesis. PMID:24112122

The competitive inhibitors of HMG-CoA reductase, popularly known as statins, exert pleiotropic effects, which result from the ability of statins to inhibit the synthesis of isoprenoids, which are fundamental for the functioning of proteins responsible for intracellular signaling. Some recent studies suggest an important role associated with the use of antineoplastic atorvastatin and rosuvastatin, the statins most widely used today. In this study, the Drosophila wing spot test was used to evaluate possible protective effects of atorvastatin and rosuvastatin against damage induced by DXR. Larvae were chronically treated with negative control (ethanol 5%), positive control (DXR 0.125?mg/mL) and five different concentrations of atorvastatin and rosuvastatin. The results demonstrated absence of a mutagenic effect for the two statins tested. The analysis of the descendants co-treated with DXR and atorvastatin/rosuvastatin revealed a modulatory effect of these statins on damage induced by DXR. This effect was verified in all concentrations tested in the descendants of the ST and HB crosses treated with rosuvastatin, and only in descendants of the HB cross treated with atorvastatin. Induction of apoptosis and antioxidant activity appear to be the main mechanisms involved in reducing the frequency of mutant spots and consequent modulation of the damage induced by DXR. PMID:25846503

In date palm (Phoenix dactylifera L. cv. Ahmar, Arecaceae), as for many monocotyledons, callogenesis is a prerequisite for the initiation of somaticembryogenesis, and requires the\\u000a presence of auxin in the medium. Immature leaf explants were cultivated in medium supplemented with either 1 or 54 ?M 1-naphtaleneacetic\\u000a acid in order to induce either rhizogenesis or callogenesis. Histological studies performed throughout the

Bortezomib is a new chemotherapeutic agent approved for the treatment of relapsed/refractory and newly diagnosed multiple myeloma. One of the major side effects of bortezomib is a peripheral length-dependent sensory axonal neuropathy and, less frequently, a small fiber neuropathy. Autonomic symptoms like postural dizziness, syncope, diarrhoea, ileus, impotence and urinary disturbances have been reported, nevertheless, autonomic neuropathy has never been characterized. We describe by means of immunofluorescence, the involvement of autonomic skin nerve fibers in three patients with small fiber neuropathy induced by bortezomib treatment. PMID:21290160

Acca sellowiana (Berg.) Burr. is a native Myrtaceae from southern Brazil and Uruguay, now the subject of a domestication and breeding program.\\u000a Biotechnological tools have been used to assist in this program. The establishment of a reliable protocol of somaticembryogenesis\\u000a has been pursued, with a view to capturing and fixing genetic gains. The rationale behind this work relies on

Theobroma cacao L., an economically important crop for developing countries, can be experimentally propagated by somaticembryogenesis. Because of their potential roles in embryogenesis, a gene candidate strategy was initiated to find gene homologues of the members of the leafy cotyledon family of transcription factors. A homologue of the leafy cotyledon1-like gene, that encodes the HAP 3 subunit of the CCAAT box-binding factor, was found in the cocoa genome (TcL1L). The translated peptide shared a high amino acid sequence identity with the homologous genes of Arabidopsis thaliana, Phaseolus coccineus and Helianthus annuus. TcL1L transcripts mainly accumulated in young and immature zygotic embryos, and, to a lesser extent, in young and immature somatic embryos. In situ hybridization specified the localization of the transcripts as being mainly in embryonic cells of young embryos, the meristematic cells of the shoot and root apex of immature embryos, and in the protoderm and epidermis of young and immature embryos, either zygotic or somatic. Non-embryogenic explants did not show TcL1L expression. Ectopic expression of the TcL1L gene could partially rescue the Arabidopsis lec1 mutant phenotype, suggesting a similarity of function in zygotic embryogenesis. PMID:18094994

After the hope and controversy brought by embryonic stem cells two decades ago for regenerative medicine, a new turn has been taken in pluripotent cells research when, in 2006, Yamanaka's group reported the reprogramming of fibroblasts to pluripotent cells with the transfection of only four transcription factors. Since then many researchers have managed to reprogram somatic cells from diverse origins into pluripotent cells, though the cellular and genetic consequences of reprogramming remain largely unknown. Furthermore, it is still unclear whether induced pluripotent stem cells (iPSCs) are truly functionally equivalent to embryonic stem cells (ESCs) and if they demonstrate the same differentiation potential as ESCs. There are a large number of reprogramming experiments published so far encompassing genome-wide transcriptional profiling of the cells of origin, the iPSCs and ESCs, which are used as standards of pluripotent cells and allow us to provide here an in-depth analysis of transcriptional profiles of human and mouse cells before and after reprogramming. When compared to ESCs, iPSCs, as expected, share a common pluripotency/self-renewal network. Perhaps more importantly, they also show differences in the expression of some genes. We concentrated our efforts on the study of bivalent domain-containing genes (in ESCs) which are not expressed in ESCs, as they are supposedly important for differentiation and should possess a poised status in pluripotent cells, i.e. be ready to but not yet be expressed. We studied each iPSC line separately to estimate the quality of the reprogramming and saw a correlation of the lowest number of such genes expressed in each respective iPSC line with the stringency of the pluripotency test achieved by the line. We propose that the study of expression of bivalent domain-containing genes, which are normally silenced in ESCs, gives a valuable indication of the quality of the iPSC line, and could be used to select the best iPSC lines out of a large number of lines generated in each reprogramming experiment. PMID:20862250

Before somaticembryogenesis can be applied, the genetic fidelity of cultures needs to be determined. Problematic of tissue-cultured\\u000a woody species is the extensive evaluation time needed for assessments. The development of methods whereby plants could be\\u000a rapidly screened for potential tissue culture-derived genetic changes would be very valuable. We evaluated the applicability\\u000a of AFLP (amplified fragment length polymorphism) analysis for

Following the discovery of pluripotent stem (PS) cells such as embryonic stem (ES) and induced pluripotent stem (iPS) cells, there has been a great hope that injured tissues can be repaired by transplantation of ES/iPS-derived various specific types of cells such as neural stem cells (NSCs). Although PS cells can be induced by ectopic expression of Yamanaka's factors, it is known that several stimuli such as ischemia/hypoxia can increase the stemness of somatic cells via reprogramming. This suggests that endogenous somatic cells acquire stemness during natural regenerative processes following injury. In this study, we describe whether somatic cells are converted into pluripotent stem cells by pathological stimuli without ectopic expression of reprogramming factors based on the findings of ischemia-induced multipotent stem cells in a mouse model of cerebral infarction. PMID:25945100

Although many studies have examined the relation between a wide range of factors and quarter milk somatic cell count (qSCC), including physical characteristics of the teat and changes in teat tissue due to milking, the effect of short-term, milking-induced changes in teat dimensions on somatic cell count has not yet been investigated. To identify teat dimensions and milking-induced changes in teat dimensions associated with qSCC, we conducted a longitudinal study (n(herds)=6, n(cows)=72, n(measurements)=12). Parity, stage of lactation, teat barrel diameter, and changes in teat barrel diameter during milking were identified as factors associated with qSCC. Teats with wider barrels had higher qSCC. Negative changes in the diameter of the teat barrel during milking (i.e., thinner teats postmilking compared with premilking) were associated with lower qSCC, whereas positive changes (i.e., thicker teats postmilking compared with premilking) were associated with higher qSCC. Selection toward more optimal teat characteristics may therefore result in improved milk quality and udder health. However, a threshold might exist for the maximum reduction in teat barrel diameter below which udder health is negatively influenced. If so, changes in teat barrel diameter might serve as an indicator for suboptimal milking and incorrect choice of teatcup liner or milking machine settings and thus help improve management of the herd. PMID:23219124

It has been suggested that transposable elements can be associated with different types of genotoxic effects. For this reason it seems appropriate to outline suitable systems to detect changes in the phenotypic expression of the loci containing transposable elements, as well as those agents that induce such changes. The sex-linked white locus offers a suitable experimental system for studying such events because most of the spontaneous mutations at the white locus are the result of insertions of repeated mobile sequences, and it is easy to follow mutational changes of the locus due to the possibility of detecting even slight changes in eye color. Here we report the results obtained in different strains of Drosophila melanogaster with copia-like induced mutations at the white locus, after treatment with three alkylating agents: ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and N-nitroso-N-ethylurea (ENU). The three insertional white mutants used in this work were wa4, wbf, and wsp55, with the wa2 mutation used as control because its mutant phenotype is the result of a point mutation instead of the insertion of a DNA fragment. Our data constitute evidence that EMS, MMS, and ENU induce a clear increase in the frequencies of somatic-revertant sectors in the three strains carrying a white allele with an inserted copia-like element. For the wa2 strain, whose mutant phenotype is the result of a point mutation, only ENU at the highest concentration tested is able to induce a significant increase in the somatic reversion frequency. In addition, our results indicate that the use of D. melanogaster strains with transposable elements in the white locus is suitable for detecting genotoxic damage induced by chemicals. PMID:7698106

Experiments were performed to determine the influence of proliferation medium on the maintenance of embryogenic competence and on repetitive embryogenesis in Castanea sativa Mill. somatic embryos derived from leaf explants. Somatic embryo proliferation was carried out by both direct secondary embryogenesis and by the culture of nodular callus tissue originated from cotyledons of somatic embryos. Both systems led to the production of cotyledonary somatic embryos on Murashige and Skoog proliferation medium supplemented with 0·1 mg l1 benzyladenine and 0·1 mg l1 naphthaleneacetic acid. Carbon source and concentration had a marked influence on maturation and subsequent germination ability of chestnut somatic embryos. Plantlet conversion was achieved in embryos matured on media with 6 % sucrose, and on 3 or 6 % maltose, whereas mean shoot length, root length and leaf number of produced plants were not significantly affected by these maturation media. Overall, the best results were obtained with 3 % maltose?matured somatic embryos, giving rise to 6 % plant recovery in addition to 33 % of embryos exhibiting only shoot development. The application of a 2?month cold treatment at 4 °C to somatic embryos matured on medium with 3 % maltose was necessary for achieving plant conversion, while partial desiccation did not appear to influence this response. A total of 39 % of embryos eventually produced plants either through conversion to plantlets or indirectly through rooting of shoots. Shoots formed by somatic embryos could be excised, multiplied and rooted following the micropropagation procedures previously developed for chestnut. PMID:12763755

In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

Group 1 late embryogenesis-abundant (LEA) proteins are a subset of hydrophilins that are postulated to play important roles in protecting plant macromolecules from damage during freezing, desiccation, or osmotic stress. To better understand the putative functional roles of group 1 LEA proteins, we analyzed the structure of a group 1 LEA protein from soybean (Glycine max). Differential scanning calorimetry of the purified, recombinant protein demonstrated that the protein assumed a largely unstructured state in solution. In the presence of trifluoroethanol (50% [w/v]), the protein acquired a 30% ?-helical content, indicating that the polypeptide is highly restricted to adopt ?-helical structures. In the presence of sodium dodecyl sulfate (1% [w/v]), 8% of the polypeptide chain adopted an ?-helical structure. However, incubation with phospholipids showed no effect on the protein structure. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein existed in equilibrium between two conformational states. Ultraviolet absorption spectroscopy studies also showed that the protein became more hydrated upon heating. Furthermore, circular dichroism spectral measurements indicated that a minimum of 14% of amino acid residues existed in a solvent-exposed, left-handed extended helical or poly (l-proline)-type (PII) conformation at 20°C with the remainder of the protein being unstructured. The content of PII-like structure increased as temperature was lowered. We hypothesize that by favoring the adoption of PII structure, instead of the formation of ?-helical or ?-sheet structures, group 1 LEA proteins retain a high content of surface area available for interaction with the solvent. This feature could constitute the basis of a potential role of LEA proteins in preventing freezing, desiccation, or osmotic stress damage. PMID:11891239

A method for direct somaticembryogenesis in alfalfa (Medicago falcata) is described. The time course in the development phase has been followed for fresh weight, cell density, pH, sugar uptake and embryo number and type. The method of disrupting the explant material has also been shown to influence subsequent embryo formation.

Carrot cells were cultured under various light spectra and intensities at different times following the initiation of suspension cultures from callus. The highest intensity white and blue light treatments were inhibitory to growth and somaticembryogenesis. Red and green light were not different from dark treatments which produced the highest total number of embryoids. After extended time in culture, carrot

Psychophysical experiments show that the perception of posture is to a large degree affected by hitherto unknown graviceptors in the human trunk. By remote control subjects move themselves radially along their spinal axis over the horizontal platform of a rotating centrifuge until they feel horizontal. Normal subjects then set the centrifuge axis on average at 22-28 cm caudal of the meatus, neuromectomized subjects at 45-55 cm. Hence the mass centroid of these receptors should be situated near the last ribs. Evaluation of the residual faculties of paraplegic patients lead to the conclusion that somatic graviception is mediated by two distinctly localized inputs, the first entering the spinal cord at the 11th thoracic segment, and the second reaching the brain cranial of the 6th cervical segment, presumably via the N. phrenicus or the N. vagus. The effect of the first named input is abolished after bilateral nephrectomy. This proves that the kidneys affect gravity perception. But whether they function like statoliths or in another way cannot yet be decided. For the second input, however, the results show unequivocally that it yields gravity information through the inertia of a mass in the body. It is hypothesized that this mass may be that of the blood in the large vessels. This is corroborated by the effect of shifting blood craniad by means of positive pressure to the legs. It is inferred that the inertial forces are measured by mechanoreceptors in the structures that mechanically support the large vessels, rather than by baroreceptors. PMID:8770370

The transcriptional response to auxin is critical for root and vascular development during Arabidopsis embryogenesis. Auxin induces the degradation of AUXIN\\/INDOLE-3-ACETIC ACID (AUX\\/IAA) transcriptional repressors, freeing their binding partners, the AUXIN RESPONSE FACTOR (ARF) proteins, which can activate transcription of auxin response genes. We show that TOPLESS (TPL) can physically interact with IAA12\\/BODENLOS (IAA12\\/BDL) through an ETHYLENE RESPONSE FACTOR (ERF)-associated

The maize abscisic acid (ABA)-responsive gene rab28 has been shown to be ABA-inducible in embryos and vegetative tissues, expression being mostly restricted to vascular elements during late embryogenesis. In the course of an expressed sequence tags (ESTs) programme, we have isolated an Arabidopsis thaliana gene, Atrab28, encoding the orthologue of maize rab28. The Atrab28 cDNA is 1090 bp long, including a

In this paper, we discussed the relationship between somatization and functional somatic syndrome (FSS). The concept of somatization takes its origin from the work of Freud S who proposed the idea of conversion as a main defense mechanism. At the same period, the term somatization was introduced by Stekel W as a hypothetical process whereby a deep-seated conflict could cause a bodily disorder. After that, Alexander F developed the emotional equivalents, which had been also proposed by Freud S, into the concept of the vegetative neurosis and psychosomatic diseases. Recently, somatization tends to be defined as 'a tendency to experience and communicate somatic distress in response to psychosocial stress and to seek medical help for it' (Lipowski ZJ, 1988). So there seems to be a strong link among conversion, somatization, FSS, and somatization disorder. PMID:19768900

Non-coding RNAs (ncRNAs) are emerging as key regulators of embryogenesis. They control embryonic gene expression by several means, ranging from microRNA-induced degradation of mRNAs to long ncRNA-mediated modification of chromatin. Many aspects of embryogenesis seem to be controlled by ncRNAs, including the maternalzygotic transition, the maintenance of pluripotency, the patterning of the body axes, the specification and differentiation of cell types and the morphogenesis of organs. Drawing from several animal model systems, we describe two emerging themes for ncRNA function: promoting developmental transitions and maintaining developmental states. These examples also highlight the roles of ncRNAs in ensuring a robust commitment to one of two possible cell fates. PMID:21245830

We have identified a novel mouse Wnt gene using a cDNA differential screening procedure for retinoic-acid-induced transcripts in P19 embryonal carcinoma cells. Sequence analysis showed that this gene represents the first murine Wnt-8 (mWnt-8) gene reported to date. The expression of the mWnt-8 gene, which is rapidly induced by retinoic acid in P19 and embryonic stem cells, appears to be

Polar auxin transport provides a developmental signal for cell fate specification during somaticembryogenesis. Some members of the HD-ZIP III transcription factors participate in regulation of auxin transport, but little is known about this regulation in somaticembryogenesis. Here, four HD-ZIP III homologues from Larix leptolepis were identified and designated LaHDZ31, 32, 33 and 34. The occurrence of a miR165/166 target sequence in all four cDNA sequences indicated that they might be targets of miR165/166. Identification of the cleavage products of LaHDZ31 and LaHDZ32 in vivo confirmed that they were regulated by miRNA. Their mRNA accumulation patterns during somaticembryogenesis and the effects of 1-N-naphthylphthalamic acid (NPA) on their transcript levels and somatic embryo maturation were investigated. The results showed that the four genes had higher transcript levels at mature stages than at the proliferation stage, and that NPA treatment down-regulated the mRNA abundance of LaHDZ31, 32 and 33 at cotyledonary embryo stages, but had no effect on the mRNA abundance of LaHDZ34. We concluded that these four members of Larix HD-ZIP III family might participate in polar auxin transport and the development of somatic embryos, providing new insights into the regulatory mechanisms of somaticembryogenesis. PMID:23566830

Microsatellites were used to test genetic stability in somatic embryos (SE) of Quercus suber L. The SE were obtained by a simple somaticembryogenesis protocol: leaf explants from two adult plants (QsG0, QsG5) and from two juvenile plants (QsGM1, QsGM2) were inoculated on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid and zeatin. Calluses with primary embryogenic structures were transferred to MSWH (MS medium without growth regulators) and SE proliferated by secondary somaticembryogenesis. High morphological heterogeneity was found among cotyledonary SE. However, converted plants looked morphologically normal with well-developed rooting systems and shoots. The genetic stability of the plant material during the somaticembryogenesis process was evaluated by using six to eight nuclear microsatellites transferred from Q. myrsinifolia Blume, Q. petraea (Matts.) Liebl. and Q. robur L. Five of eight microsatellites distinguished among the genotypes analyzed, and for QsG0, QsGM1 and QsGM2, uniform microsatellite patterns were generally observed within and between SE and the respective donor genotypes. For genotype QsG5, the same pattern was observed in all samples analyzed except one, where the mutation percentage was 2.5%. We conclude that microsatellite markers can be used to assess genetic stability of clonal materials and to determine genetic stability throughout the process of somaticembryogenesis. The simple somaticembryogenesis protocol described has potential for the commercial propagation of Q. suber because it results in a low percentage of mutations. PMID:16740490

Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced into an oocyte from which its genetic material has been removed by a process called enucleation. In mammals, the reconstructed embryo is artificially induced to initiate embryonic development (activation). The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear reprogramming and involves an important change of cell fate, by which the somatic cell nucleus becomes capable of generating all the cell types required for the formation of a new individual, including extraembryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring genetically identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the nuclear transplantation technique for research or commercial purposes. PMID:20336522

Somatic cloning in cattle is associated with impaired embryo development, caused by inappropriate epigenetic reprogramming during embryogenesis; however, there is a paucity of data regarding gene expression at the critical elongation and peri-implantation stages. The objective of the present study was to identify genes differentially expressed in bovine cloned embryos at Day 17 of development (Day 0=day of nucleus transfer

Intact somatic embryos were obtained from an elite maize inbred line Y423, bred in our laboratory. Using 13-day immature embryos after self-pollination as explants, and after 4-5 times subculture, a large number of somatic embryos were detected on the surface of the embryonic calli on the medium. The intact somatic embryos were transferred into the differential medium, where the plantlets regenerated with shoots and roots forming simultaneously. Histological analysis and scanning electron micrographs confirmed the different developmental stages of somaticembryogenesis, including globular-shaped embryo, pear-shaped embryo, scutiform embryo, and mature embryo. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used for comparative transcript profiling between embryogenic and non-embryogenic calli of a new elite maize inbred line Y423 during somaticembryogenesis. Differentially expressed genes were cloned and sequenced. Gene Ontology analysis of 117 candidate genes indicated their involvement in cellular component, biological process and molecular function. Nine of the candidate genes were selected. The changes in their expression levels during embryo induction and regeneration were analyzed in detail using quantitative real-time PCR. Two full-length cDNA sequences, encoding ZmSUF4 (suppressor of fir 4-like protein) and ZmDRP3A (dynamin-related protein), were cloned successfully from intact somatic embryos of the elite inbred maize line Y423. Here, a procedure for maize plant regeneration from somatic embryos is described. Additionally, the possible roles of some of these genes during the somaticembryogenesis has been discussed. This study is a systematic analysis of the cellular and molecular mechanism during the formation of intact somatic embryos in maize. PMID:25931320

The effects of exercise on skeletal muscle are mediated by a coupling between muscle electrical activity and gene expression. Several activity correlates, such as intracellular Ca2+, hypoxia and metabolites like free fatty acids (FFAs), might initiate signalling pathways regulating fibre-type-specific genes. FFAs can be sensed by lipid-dependent transcription factors of the peroxisome proliferator-activated receptor (PPAR) family. We found that the mRNA for the predominant muscle isoform, PPAR?, was three-fold higher in the slow/oxidative soleus compared to the fast/glycolytic extensor digitorum longus (EDL) muscle. In histological sections of the soleus, the most oxidative fibres display the highest levels of PPAR? protein. When the soleus muscle was stimulated electrically by a pattern mimicking fast/glycolytic IIb motor units, the mRNA level of PPAR? was reduced to less than half within 24 h. In the EDL, a three-fold increase was observed after slow type I-like electrical stimulation. When a constitutively active form of PPAR? was overexpressed for 14 days in normally active adult fibres after somatic gene transfer, the number of I/IIa hybrids in the EDL more than tripled, IIa fibres increased from 14% to 25%, and IIb fibres decreased from 55% to 45%. The level of succinate dehydrogenase activity increased and size decreased, also when compared to normal fibres of the same type. Thus PPAR? can change myosin heavy chain, oxidative enzymes and size locally in muscle cells in the absence of general exercise. Previous studies on PPAR? in muscle have been performed in transgenic animals where the transgene has been present during muscle development. Our data suggest that PPAR? can mediate activity effects acutely in pre-existing adult fibres, and thus is an important link in excitationtranscription coupling. PMID:17463039

This study provides a physiological analysis of somaticembryogenesis in four elite cultivars of date palms: Ahmar, Amsekhsi, Tijib, and Amaside, from the initial callogenesis to establishment and proliferation of embryogenic suspension cultures. Somatic embryos development and in vitro plants rooting were also studied. For each step, auxins and cytokinins concentrations were optimised. The primary callogenesis from leaf explants of seedlings appeared highly dependent on genotype. Ahmar (80%) and Amsekhsi (76%) appeared highly callogenic, whereas Tijib (10%) and Amaside (2%) produced low amounts of calluses. 2,4-Dichlorophenoxyacetic acid appeared favorable to the induction of primary callogenesis and its effect was enhanced by the addition of benzyl adenine or adenine sulfate. Secondary friable calli obtained from chopped granular calli were used to initiate embryogenic cell suspensions in media supplied with 2,4-dichlorophenoxyacetic acid. Suspension cultures showed a growth rate of fourfold after four subcultures in presence of 2,4-dichlorophenoxyacetic acid 2?mg/L. Our results showed that a seven-day transitory treatment with benzyl adenine 0,5?mg/L was necessary to optimize embryos development. Naphthalene acetic acid induced the development of primary orthogravitropic roots during embryos germination. The comparison with cytofluorometry of nuclear DNA amounts showed no significant difference in ploidy level between regenerated plants and seedlings. PMID:22629211

Free speech of subjects with somatization and paranoia was analyzed to identify and compare self-concept dimensions reflected in their lexical choices. The somatization disorder group conveyed a sense of negativism, distress, and preoccupation with an uncertain self-identity. The paranoid patients portrayed an artificially positive, grandiose

Feijoa (Acca sellowiana, Myrtaceae), a native fruit species from southern Brazil and northern Uruguay, is considered to constitute a reference system\\u000a for somaticembryogenesis in woody dicots. This in vitro regenerative pathway is an efficient micropropagation method, and\\u000a a suitable model system for studies in plant developmental physiology. This study attempts to detect and identify proteins\\u000a that are expressed during

Somatization is the psychological mechanism whereby psychological distress is expressed in the form of physical symptoms. The psychological distress in somatization is most commonly caused by a mood disorder that threatens mental stability. Conversion disorder occurs when the somatic presentation involves any aspect of the central nervous system over which voluntary control is exercised. Conversion reactions represent fixed ideas about neurologic malfunction that are consciously enacted, resulting in psychogenic neurologic deficits. Treatment is complex and lengthy; it includes recovery of neurologic function aided by narcoanalysis and identification and treatment of the primary psychiatric disorder, usually a mood disorder. PMID:15101499

The growth factors that drive the division and differentiation of stem cells during early human embryogenesis are unknown. The secretion of endorphins, progesterone (P4), human chorionic gonadotropin, 17?-estradiol, and gonadotropin-releasing hormone by trophoblasts that lie adjacent to the embryoblast in the blastocyst suggests that these pregnancy-associated factors may directly signal the growth and development of the embryoblast. To test this hypothesis, we treated embryoblast-derived human embryonic stem cells (hESCs) with ICI 174,864, a ?-opioid receptor antagonist, and RU-486 (mifepristone), a P4 receptor competitive antagonist. Both antagonists potently inhibited the differentiation of hESC into embryoid bodies, an in vitro structure akin to the blastocyst containing all three germ layers. Furthermore, these agents prevented the differentiation of hESC aggregates into columnar neuroectodermal cells and their organization into neural tube-like rosettes as determined morphologically. Immunoblot analyses confirmed the obligatory role of these hormones; both antagonists inhibited nestin expression, an early marker of neural precursor cells normally detected during rosette formation. Conversely, addition of P4 to hESC aggregates induced nestin expression and the formation of neuroectodermal rosettes. These results demonstrate that trophoblast-associated hormones induce blastulation and neurulation during early human embryogenesis. PMID:18803462

Background Piwi proteins are essential for germ line development, stem cell maintenance, and more recently found to function in epigenetic and somatic gene regulation. In the sea urchin Strongylocentrotus purpuratus, two Piwi proteins, Seawi and Piwi-like1, have been identified, yet their functional contributions have not been reported. Result Here we found that Seawi protein was localized uniformly in the early embryo and then became enriched in the primordial germ cells (PGCs) (the small micromere lineage) from blastula stage and thereafter. Morpholino knockdown of Sp-seawi diminished PGC-specific localization of Seawi proteins, and altered expression of other germ line markers such as Vasa and Gustavus, but had no effect on Nanos. Further, Seawi knockdown transiently resulted in Vasa positive cell proliferation in the right coelomic pouch that appear to be derived from the small micromere lineage, yet they quickly disappeared with an indication of apoptosis by larval stage. Severe Seawi knockdown resulted in an increased number of apoptotic cells in the entire gut area. Piwi proteins appear to regulate PGC proliferation perhaps through control of Vasa accumulation. In this organism, Piwi is likely regulating mRNAs, not just transposons, and is potentially functioning both inside and outside of the germ line during embryogenesis. PMID:24218044

Somatic mutations of the KIT gene have been reported in mast cell diseases and gastrointestinal stromal tumours. Recently, they have also been found in mediastinal and testicular germ cell tumours (TGCTs), particularly in cases with bilateral disease. We screened the KIT coding sequence (except exon 1) for germline mutations in 240 pedigrees with two or more cases of TGCT. No germline mutations were found. Exons 10, 11 and 17 of KIT were examined for somatic mutations in 123 TGCT from 93 multiple-case testicular cancer families. Five somatic mutations were identified; four were missense amino-acid substitutions in exon 17 and one was a 12 bp in-frame deletion in exon 11. Two of seven TGCT from cases with bilateral disease carried KIT mutations compared with three out of 116 unilateral cases (P=0.026). The results indicate that somatic KIT mutations are implicated in the development of a minority of familial as well as sporadic TGCT. They also lend support to the hypothesis that KIT mutations primarily take place during embryogenesis such that primordial germ cells with KIT mutations are distributed to both testes. PMID:15150569

Background: Chlorinated phosphate esters (CPEs) are widely used as additive flame retardants for low-density polyurethane foams and have frequently been detected at elevated concentrations within indoor environmental media. Objectives: To begin characterizing the potential toxicity of CPEs on early vertebrate development, we examined the developmental toxicity of four CPEs used in polyurethane foam: tris(1,3-dichloro-2-propyl) phosphate (TDCPP), tris(2-chloroethyl) phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCPP), and 2,2-bis(chloromethyl)propane-1,3-diyl tetrakis(2-chlorethyl) bis(phosphate) (V6). Methods: Using zebrafish as a model for vertebrate embryogenesis, we first screened the potential teratogenic effects of TDCPP, TCEP, TCPP, and V6 using a developmental toxicity assay. Based on these results, we focused on identification of susceptible windows of developmental TDCPP exposure as well as evaluation of uptake and elimination of TDCPP and bis(1,3-dichloro-2-propyl)phosphate (BDCPP, the primary metabolite) within whole embryos. Finally, because TDCPP-specific genotoxicity assays have, for the most part, been negative in vivo and because zygotic genome remethylation is a key biological event during cleavage, we investigated whether TDCPP altered the status of zygotic genome methylation during early zebrafish embryogenesis. Results: Overall, our findings suggest that the cleavage period during zebrafish embryogenesis is susceptible to TDCPP-induced delays in remethylation of the zygotic genome, a mechanism that may be associated with enhanced developmental toxicity following initiation of TDCPP exposure at the start of cleavage. Conclusions: Our results suggest that further research is needed to better understand the effects of a widely used and detected CPE within susceptible windows of early vertebrate development. PMID:23017583

Mouse tumors induced by gamma radiation are a useful model system for oncogenesis. DNA from such tumors contains an activated K-ras oncogene that can transform NIH 3T3 cells. This report describes the cloning of a fragment of the mouse K-ras oncogene containing the first exon from both a transformant in rat-2 cells and the brain of the same mouse that developed the tumor. Hybrid constructs containing one of the two pieces were made and only the plasmid including the first exon from the transformant gave rise to foci in NIH 3T3 cells. There was only a single base difference (G----A) in the exonic sequence, which changed glycine to aspartic acid in the transformant. By use of a synthetic oligonucleotide the presence of the mutation was demonstrated in the original tumor, ruling out modifications during DNA-mediated gene transfer and indicating that the alteration was present in the thymic lymphoma but absent from other nonmalignant tissue. The results are compatible with gamma radiation being a source of point mutations.

A significant work on callus induction and somaticembryogenesis was realized for Hibiscus sabdariffa. Two genotypes (Hibiscus sabdariffa and Hibiscus sabdariffa var. altissima) two sugars (sucrose and glucose) and three concentrations (1 %, 2%, 3%) of each sugar, 3 explant types (root, hypocotyl, c...

1 Left-Right Asymmetry in Animal Embryogenesis Michael Levin Cell Biology dept. Bldg. C1, rm. 403 of a given type) differences between the left and right sides of an animal's morphology. This specifically of an organism looking the same to some level of detail on either side of a symmetry line). Animal body- plans

Comparative analysis of early embryogenesis indicates that considerable differences exist among nematode species. To better understand to what extent the well-studied development of Caenorhabditis elegans is representative for nematodes in general, we extended our earlier studies to other families of this phylum. Here we report our findings on seven species of Plectidae. We found that Plectidae embryos share a number

Differentiating calli derived from rice (Oryza sativa L.) microspores were examined histologically. Shoot and root meristems were observed to be arising by both organogenesis as well as embryogenesis. Embryoid attachment to callus (as well as other embryoids) was at the scutellum adjacent to the mesocotyl and radicle. These observations could be interpreted as an indication of the totipotent plasticity of

The transcriptional response to auxin is critical for root and vascular development during Arabidopsis embryogenesis. Auxin induces the degradation of AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) transcriptional repressors, freeing their binding partners, the AUXIN RESPONSE FACTOR (ARF) proteins, which can activate transcription of auxin response genes. We show that TOPLESS (TPL) can physically interact with IAA12/BODENLOS (IAA12/BDL) through an ETHYLENE RESPONSE FACTOR (ERF)-associated amphiphilic repression (EAR) motif. TPL can repress transcription in vivo and is required for IAA12/BDL repressive activity. In addition, tpl-1 can suppress the patterning defects of the bdl-1 mutant. Direct interaction between TPL and ARF5/MONOPTEROS, which is regulated by IAA12/BDL, results in a loss-of-function arf5/mp phenotype. These observations show that TPL is a transcriptional co-repressor and further our understanding of how auxin regulates transcription during plant development. PMID:18258861

Cloning by nuclear transfer from adult somatic cells is a remarkable demonstration of developmental plasticity. When a nucleus is placed in oocyte cytoplasm, the changes in chromatin structure that govern differentiation can be reversed, and the nucleus can be made to control development to term.

Richard Shusterman's "Body Consciousness" aims at formulating a theory of somaesthetics and somatic experience. There has indeed been a growing interest in the role of the body in experience. Shusterman examines the arguments of six important writers who have been influential in this discussion. The emphasis on the body is natural for a

Appropriate development of stratified, squamous, keratinizing epithelia, such as the epidermis and oral epithelia, generates an outer protective permeability barrier that prevents water loss, entry of toxins, and microbial invasion. During embryogenesis, the immature ectoderm initially consists of a single layer of undifferentiated, cuboidal epithelial cells that stratifies to produce an outer layer of flattened periderm cells of unknown function. Here, we determined that periderm cells form in a distinct pattern early in embryogenesis, exhibit highly polarized expression of adhesion complexes, and are shed from the outer surface of the embryo late in development. Mice carrying loss-of-function mutations in the genes encoding IFN regulatory factor 6 (IRF6), I?B kinase-? (IKK?), and stratifin (SFN) exhibit abnormal epidermal development, and we determined that mutant animals exhibit dysfunctional periderm formation, resulting in abnormal intracellular adhesions. Furthermore, tissue from a fetus with cocoon syndrome, a lethal disorder that results from a nonsense mutation in IKKA, revealed an absence of periderm. Together, these data indicate that periderm plays a transient but fundamental role during embryogenesis by acting as a protective barrier that prevents pathological adhesion between immature, adhesion-competent epithelia. Furthermore, this study suggests that failure of periderm formation underlies a series of devastating birth defects, including popliteal pterygium syndrome, cocoon syndrome, and Bartsocas-Papas syndrome. PMID:25133425

Introduction Unrestricted somatic stem cells (USSC) derived from umbilical cord blood are an attractive alternative to human embryonic\\u000a stem cells (hESC) for cellular therapy. USSC are capable of forming cells representative of all three germ line layers. The\\u000a aim of this study was to determine the potential of USSC to form definitive endoderm following induction with Activin A, a\\u000a protein known

The wing Somatic Mutation And Recombination Test (SMART) in Drosophila melanogaster was used to study the modulating action of vanillin (VA) in combination with the alkylating agents mitomycin C (MMC), methylmethanesulphonate (MMS) and the bifunctional nitrogen mustard (HN2). Two types of treatments with VA and each of the three genotoxins were performed: chronic co-treatments of three-day-old larvae of the standard

Wolbachia are required for filarial nematode survival and fertility and contribute to the immune responses associated with human filarial diseases. Here we developed whole-mount immunofluorescence techniques to characterize Wolbachia somatic and germline transmission patterns and tissue distribution in Brugia malayi, a nematode responsible for lymphatic filariasis. In the initial embryonic divisions, Wolbachia segregate asymmetrically such that they occupy only a small subset of cells in the developing embryo, facilitating their concentration in the adult hypodermal chords and female germline. Wolbachia are not found in male reproductive tissues and the absence of Wolbachia from embryonic germline precursors in half of the embryos indicates Wolbachia loss from the male germline may occur in early embryogenesis. Wolbachia rely on fusion of hypodermal cells to populate adult chords. Finally, we detect Wolbachia in the secretory canal lumen suggesting living worms may release bacteria and/or their products into their host. PMID:20689574

Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis. PMID:24423870

\\u000a Nuclear and somatic cell genetic reprogramming has seen a huge improvement during the past 10 years since the cloning of the\\u000a first mammal, Dolly the sheep. In this chapter we will summarise the advancement in nuclear and cell reprogramming by cell\\u000a fusion, using amphibian eggs or egg extracts, with cell extracts, with synthetic molecules, or by induced expression of specific

Somatization disorder (SD), a chronic psychiatric illness that affects about 1% of adult women, is characterized by multiple somatic complaints. It should be suspected in any woman who presents with a vague or complicated history; unaccountable non-responsiveness to therapy; dramatic, seductive or demanding personality style; family history of personality disorder; sexual abuse as a child; substance abuse; or depression with atypical features. Its cause is unknown, although both genetic and environmental factors have been implicated. At follow-up, patients with SD continue to have somatic symptoms, but many improve with therapy. Nearly two thirds of patients with SD attempt suicide, but few complete it; however, completions may be more common than formerly realized. There is no specific treatment for SD, but management can be organized around the following ABCs: Accommodate initially to forge rapport; Behavior modification (ignore symptoms, praise for improved behavior); Confrontation later about effects of behavior style; Decrease drugs gradually, with praise for reduction; Educate about course and meaning of illness; Family involvement to give information and help with treatment; Guilt should be assuaged in physicians, who may blame themselves when patients do not improve; Hospitalize (closed psychiatric unit) only for serious suicide risk, substance abuse, or other extreme behavior; and Intercurrent depression should be treated conservatively. PMID:2209356

BACKGROUND: The actin cytoskeleton plays critical roles in early development in Caenorhabditis elegans. To further understand the complex roles of actin in early embryogenesis we use RNAi and in vivo imaging of filamentous actin (F-actin) dynamics. RESULTS: Using RNAi, we found processes that are differentially sensitive to levels of actin during early embryogenesis. Mild actin depletion shows defects in cortical

This is a lab exercise geared toward first-year undergraduate biology majors, where they get to view early embryogenesis in a live animal. In this exercise students will prepare slides if live C. elegans embryos, find one- or two-cell stage embryos, and observe cleavage stage of embryogenesis over the course of 30 minutes.

Generating gametes from pluripotent stem cells (PSCs) has many scientific justifications and several biomedical rationales. Here, we consider several strategies for deriving gametes from PSCs from mice and primates (human and non-human) and their anticipated strengths, challenges and limitations. Although the Weismann barrier, which separates the mortal somatic cell lineages from the potentially immortal germline, has long existed, breakthroughs first in mice and now in humans are artificially creating germ cells from somatic cells. Spermatozoa with full reproductive viability establishing multiple generations of seemingly normal offspring have been reported in mice and, in humans, haploid spermatids with correct parent-of-origin imprints have been obtained. Similar progress with making oocytes has been published using mouse PSCs differentiated in vitro into primordial germ cells, which are then cultured after xenografting reconstructed artificial ovaries. Progress in making human oocytes artificially is proving challenging. The usefulness of these artificial gametes, from assessing environmental exposure toxicity to optimising medical treatments to prevent negative off-target effects on fertility, may prove invaluable, as may basic discoveries on the fundamental mechanisms of gametogenesis. PMID:25472048

Tef (Eragrostis tef) provides a major source of human nutrition in the Horn of Africa, but biotechnology has had little impact on its improvement to date. Here, we report the elaboration of an in vitro regeneration protocol, based on the use of immature zygotic embryos as explant. Explant size was an important determinant of in vitro regeneration efficiency, as was the formulation of the culture medium. Optimal results were obtained by culturing 0.2-0.35?mm embryo explants on a medium containing KBP minerals, 9.2-13.8??M 2,4-dichlorophenoxyacetic acid, 6?mM glutamine, and 0.5% Phytagel. Although this protocol was effective for both the improved cultivar "DZ-01-196" and the landrace "Fesho", the former produced consistently more embryogenic tissue and a higher number of regenerants. An average of more than 2,800 shoots could be obtained from each "DZ-01-196" explant after 12 weeks of in vitro culture. These shoots readily formed roots, and plantlets transferred to soil were able to develop into morphologically normal, fertile plants. This regeneration and multiplication system should allow for the application of a range of biotechnological methods to tef. PMID:22028975

The mature seeds, mesocotyls, and young leaf tips of Elymus sibiricus L. cv. chuancao No. 2 were cultured on Murashige and Skoog (MS) medium supplemented with 5.0 mg\\/L 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.05 mg\\/L kinetin in the dark at 26°C, the calluses were produced. The rate of callus regeneration depended on the explants\\u000a source and plant growth regulators. Plants regenerated from whitish-yellow-coloured

Several economically important tree species belong to the genus Pinus\\u000a and many of them form the ecological base of forest ecosystems. Pine wood is an important raw material\\u000a for the forest industry and many of the pine species have been involved in conventional tree improvement\\u000a programmes. A lot of effort has been made in the development of vegetative propagation methods,\\u000a especially

Black locust (Robinia pseudoacacia L.) immature seeds of different developmental stages were tested for the ability to initiate embryogenic cultures. Best results (average of 12% embryogenic cultures) were obtained when seeds collected 23 weeks post-anthesis were cultured for 3 weeks on modified Finer and Nagasawa medium containing 2,4-D (4590 µM) and BA (2.2 µM) and then transferred to the same

The crop species Olea europaea L. (olive tree) is of great economic importance in the Mediterranean region. Hence, many efforts have been done in the last\\u000a decades to propagate this commercially valuable species by in vitro methods. On the other hand, the lesser known Olea maderensis (Lowe) Rivas Mart. & Del Arco which is a native species of the Madeira

A carrot cell culture seeded in Petri dishes in the United States and transported to the USSR was subjected to weightlessness for 20 days during the flight of Kosmos 782. The controls were cultures placed on a centrifuge (1 g) inside the satellite and cultures left on ground in the U.S.S.R. and the United States. A count of structures in the dishes after the flight showed that the number of developing embryonic structures and the extent of their differentiation in weightlessness did not reliably differ from the number and extent of differentiation in structures developed on the ground. Structures with long roots developed in weightlessness. Analysis of the root zones showed that these roots differed by the increased size of the zone of differentiated cells. The increased size of the zones of differentiated cells can indicate earlier development of embryonic structures.

and the sensitivities of the camera result in different images. While a setting for laser and camera may produce a large expression below the minimum that can be captured by the instruments. The laser strength and the sensitivity of the camera to light can be adjusted by the operator to find a `best' picture, one where most of the spots

strengths and the sensitivities of the camera result in different images. While a setting for laser. The laser strength and the sensitivity of the camera to light can be adjusted by the operator to find the strength of the laser and the sensitivity of the camera used to record expression levels and propose

Tuberous roots of Chlorophytum borivilianum Sant. et Fernand. which are a source of steroidal saponins, possess immunomodulatory, adaptogenic, aphrodisiac, antipyretic,\\u000a diuretic, hemostatic and anti-tumour properties. Poor seed setting and germination and slow growth in conventional vegetative\\u000a propagation are major constraints in the large-scale cultivation of this commercially important medicinal plant. In the present\\u000a study, a procedure for in vitro propagation

Cultures of preglobular stage proembryos (PGSPs) were initiated from mechanically wounded mature zygotic embryos of carrot, Daucus carota, on a hormone-free, semisolid medium. These PGSPs have been maintained and multiplied for extended periods without their progression into later embryo stages on the same hormone-free medium containing 1 mM NH4+ as the sole nitrogen source. Sustained maintenance of cultures comprised exclusively of PGSPs was dependent on medium pH throughout the culture period. Best growth and multiplication of PGSP cultures occurred when the pH of unbuffered, hormone-free medium fell from 4.5 to 4 over a 2-week period or when buffered medium was titrated to pH 4. If the hormone-free medium was buffered to sustain a pH at or above 4.5, PGSPs developed into later embryo stages. Maintenance with continuous multiplication of PGSPs occurred equally well on medium containing NH4+ or NH4+ and NO3-, but growth was poor with NO3- alone. Additional observations on the effects of medium components such as various nitrogen sources and levels, sucrose concentration, semisolid supports, type of buffer, borate concentration, activated charcoal, and initial pH that permit optimum maintenance of the PGSPs or foster their continued developmental progression into mature embryos and plantlets are reported. The influence of the pH of the hormone-free medium as a determinant in maintaining cultures as PGSPs or allowing their continued embryonic development are unequivocally demonstrated by gross morphology, scanning electron microscopy, and histological preparations.

Protoplasts derived from hypocotyls of seedlings grown on half-strength MS medium containing 1% sucrose were cultured at a density of 5×104 ml-1 in Kao's medium supplemented with 1.0 mgl-12,4-D, 0.1 mgl-1 NAA and 0.5 mgl-1 zeatin riboside. After three days of culture in darkness at 25±1°C, cultures were transferred to light (70 µEm-2s-1) in a 16\\/8 h ligø ht\\/dark cycle.

Somatic cells are an important component naturally present in milk, and somatic cell count is used as an indicator of udder health and milk quality. The role of somatic cells in dairy processes and products is ill-defined in most studies because the role of these cells combines also the concomitance of physicochemical modifications of milk, bacterial count, and the udder inflammation in the presence of high somatic cell count. The aim of this review is to focus on the role of somatic cells themselves and of endogenous enzymes from somatic cells in milk, in dairy transformation processes, and in characteristics of final products overcoming biases due to other factors. The immune function of somatic cells in the udder defense and their protective role in milk will be primarily considered. Different characteristics of milk induced by various somatic cell counts, types, and their endogenous enzymes influencing directly the technological properties of milk and the final quality of dairy products will be discussed as well. By comparing methods used in other studies and eliminating biases due to other factors not considered in these studies, a new approach has been suggested to evaluate the effective role of somatic cells on dairy processes and products. In addition, this new approach allows the characterization of somatic cells and their endogenous enzymes and, in future research, will allow the clarification of mechanisms involved in the release of these components from somatic cells during dairy processes, particularly in cheese technologies. PMID:25309683

Glycine max (soybean) is the only known higher plant with a definitely established occurrence of somatic crossing over. This material lends itself to the analysis of somatic crossing over, gross chromosomal aberrations and mu- tations, all of which may be induced by the same treatment of the mutagen given to seeds. This is made possible because gene Y,, for chlorophyll

Plants are exposed to different external conditions that affect growth, development, and productivity. Water deficit is one of these adverse conditions caused by drought, salinity, and extreme temperatures. Plants have developed different responses to prevent, ameliorate or repair the damage inflicted by these stressful environments. One of these responses is the activation of a set of genes encoding a group of hydrophilic proteins that typically accumulate to high levels during seed dehydration, at the last stage of embryogenesis, hence named Late Embryogenesis Abundant (LEA) proteins. LEA proteins also accumulate in response to water limitation in vegetative tissues, and have been classified in seven groups based on their amino acid sequence similarity and on the presence of distinctive conserved motifs. These proteins are widely distributed in the plant kingdom, from ferns to angiosperms, suggesting a relevant role in the plant response to this unfavorable environmental condition. In this review, we analyzed the LEA proteins from those legumes whose complete genomes have been sequenced such as Phaseolus vulgaris, Glycine max, Medicago truncatula, Lotus japonicus, Cajanus cajan, and Cicer arietinum. Considering their distinctive motifs, LEA proteins from the different groups were identified, and their sequence analysis allowed the recognition of novel legume specific motifs. Moreover, we compile their transcript accumulation patterns based on publicly available data. In spite of the limited information on these proteins in legumes, the analysis and data compiled here confirm the high correlation between their accumulation and water deficit, reinforcing their functional relevance under this detrimental conditions. PMID:23805145

Summary Somatic embryos of pineapple guava (Feijoa sellowiana Berg, Myrtaceae) were induced particularly well from the adaxial face of the cotyledons of zygotic embryos cultured on MS medium containing 1.0 mg\\/l 2,4-D and 0.3 M sucrose. Somatic embryos were never obtained from globular and heart-shaped zygotic embryos and embryos at the torpedo stage produced somatic embryos at lower frequencies than

The maize abscisic acid (ABA)-responsive gene rab28 has been shown to be ABA-inducible in embryos and vegetative tissues, expression being mostly restricted to vascular elements during late embryogenesis. In the course of an expressed sequence tags (ESTs) programme, we have isolated an Arabidopsis thaliana gene, Atrab28, encoding the orthologue of maize rab28. The Atrab28 cDNA is 1090 bp long, including a poly(A)+ stretch, and encodes a polypeptide of 262 amino acids. Atrab28 antibody against the recombinant protein recognizes a polipeptide of about 30 kDa and pI 6, in close agreement with the predicted molecular mass and pI. As for maize rab28, expression studies with Atrab28 revealed high specificity for embryo tissues, transcription being stimulated by the transcriptional activator abi3. In contrast, Atrab28 was not induced in vegetative tissues by ABA, osmotic stress or dehydration. The expression of Atrab28 mRNA and the accumulation of Atrab28 protein was largely restricted to provascular tissues of mature embryos and in the seed coat outer tegument and embryo and silique epidermis, as revealed by in situ hybridization and immunocytochemistry with anti-Atrab28 antibodies. PMID:10412913

Conclusions During the last decade, a substantial body of knowledge has been obtained on the generation of somatic diversification of the B cell repertoire, especially with regard to differentiation and selection of B cells in specialized microenvironments such as GCs and GALTs which are similar to each other morphologically and physiologically. Although the mechanisms for somatic diversification remain unclear, with

Brassica napus cultivar Westar is non-embryogenic under all standard protocols for induction of microspore embryogenesis; however, the rare embryos produced in Westar microspore cultures, induced with added brassinosteroids, were found to develop into heritably stable embryogenic lines after chromosome doubling. One of the Westar-derived doubled haploid (DH) lines, DH-2, produced up to 30% the number of embryos as the highly embryogenic B. napus line, Topas DH4079. Expression analysis of marker genes for embryogenesis in Westar and the derived DH-2 line, using real-time reverse transcription-PCR, revealed that the timely expression of embryogenesis-related genes such as LEAFY COTYLEDON1 (LEC1), LEC2, ABSCISIC ACID INSENSITIVE3, and BABY BOOM1, and an accompanying down-regulation of pollen-related transcripts, were associated with commitment to embryo development in Brassica microspores. Microarray comparisons of 7?d cultures of Westar and Westar DH-2, using a B. napus seed-focused cDNA array (10?642 unigenes), identified highly expressed genes related to protein synthesis, translation, and response to stimulus (Gene Ontology) in the embryogenic DH-2 microspore-derived cell cultures. In contrast, transcripts for pollen-expressed genes were predominant in the recalcitrant Westar microspores. Besides being embryogenic, DH-2 plants showed alterations in morphology and architecture as compared with Westar, for example epinastic leaves, non-abscised petals, pale flower colour, and longer lateral branches. Auxin, cytokinin, and abscisic acid (ABA) profiles in young leaves, mature leaves, and inflorescences of Westar and DH-2 revealed no significant differences that could account for the alterations in embryogenic potential or phenotype. Various mechanisms accounting for the increased capacity for embryogenesis in Westar-derived DH lines are considered. PMID:18552352

Bipolaris oryzae is a phytopathogenic fungus causing a brown spot disease in rice, and produces substance that strongly perturbs motility and membrane integrities of boar spermatozoa. The substance was isolated from the liquid culture of the fungal strain using extraction and a multi-step semi-preparative HPLC procedures. Based on the results of mass spectrometric and 2D NMR techniques, the bioactive molecule was identified as ophiobolin A, a previously described sesterterpene-type compound. The purified ophiobolin A exhibited strong motility inhibition and viability reduction on boar spermatozoa. Furthermore, it damaged the sperm mitochondria significantly at sublethal concentration by the dissipation of transmembrane potential in the mitochondrial inner membrane, while the plasma membrane permeability barrier remained intact. The study demonstrated that the cytotoxicity of ophiobolin A toward somatic cell lines is higher by 12 orders of magnitude compared to other mitochondriotoxic mycotoxins, and towards sperm cells unique by replacing the progressive motility by shivering tail beating at low exposure concentration. PMID:25251540

Conversion of somatic cells to pluripotency by defined factors is a long and complex process that yields embryonic stem cell-like cells that vary in their developmental potential. To improve the quality of resulting induced pluripotent stem cells (iPSCs), which is important for potential therapeutic applications, and to address fundamental questions about control of cell identity, molecular mechanisms of the reprogramming process must be understood. Here we discuss recent discoveries regarding the role of reprogramming factors in remodeling the genome, including new insights into the function of c-Myc, and describe the different phases, markers and emerging models of reprogramming. PMID:23681063

Instability and enlargement of a CAG repeat region at the beginning of the huntingtin gene (IT-15) has been linked with Huntington`s disease. The CAG repeat size shows a highly significant correlation with age-of-onset of clinicial features in individuals with 40 or more repeats who have Huntington disease. The clinical status of nonsymptomatic individuals with 30 to 39 CAG repeats is considered ambiguous. In order to define more carefully the nature of the HD expansion instability, we examined patients in our HD population using a discriminating fluorescence-based PCR approach. The degree of somatic mutation increases with both earlier age of onset and the size of the inherited allele. A single prominent band one repeat larger than the index peak was typical in individuals with 40-41 CAG repeats. Three to four larger bands are typically discerned in individuals with 50 or more repeats. In an extreme example, an individual with approximately 95 repeats had at least 8 prominent bands. Plotting the degree of somatic mutation relative to the size of the HD allele shows somatic mutation activity increases with size. By this approach 40-60% of the alleles in a 40-41 CAG repeat HD loci is represented in the primary allele. In contrast, the primary allele represents a relatively minor proportion of the total alleles for expansions greater than 50 CAG repeats (10-20%). The limited range of somatic mutation suggest that the instability is restricted to very early stages of embryogenesis before tissue development diverges or that persistent somatic instability occurs at a slow rate. Therefore, the properties of somatic instability in Huntington`s disease have aspects that are both in common but also different from that found in other trinucleotide repeat expanding diseases such as myotonic muscular dystrophy and fragile X syndrome.

The occurrence and significance of changes in cell wall components and signalling molecules has been investigated during early microspore embryogenesis in cork oak (Quercus suber L.) in relation to cell proliferation and cell differentiation. Microspore embryogenesis has been induced in in vitro anther cultures of Q. suber by the application of a stress treatment of 33 degrees C. After the treatment, microspores at the responsive developmental stage of vacuolate microspore switched towards proliferation and the embryogenesis pathway to further produce haploid plantlets. Ultrastructural and immunocytochemical analysis revealed changes in cell organisation after induction at different developmental stages, the cellular features displayed being in relation to the activation of proliferative activity and the beginning of differentiation in young and late proembryos. Immunogold labelling with JIM5 and JIM7 antibodies showed a different presence of pectin and level of its esterification in cell walls at different developmental stages. Non-esterified pectins were found in higher proportions in cells of late proembryos, suggesting that pectin de-esterification could be related to the beginning of differentiation. The presence and subcellular distribution of Erk 1/2 MAPK homologues have been investigated by immunoblotting, immunofluorescence and immunogold labelling. The results showed an increase in the expression of these proteins with a high presence in the nucleus, during early microspore proembryos development. The reported changes during early microspore embryogenesis are modulated in relation to proliferation and differentiation events. These findings provided new evidences for a role of MAPK signalling pathways in early microspore embryogenesis, specifically in proliferation, and would confer information for the cell fate and the direction of the cell development. PMID:15346811

The use of doubled haploids in onion breeding is limited due to the low gynogenesis efficiency of this species. Gynogenesis capacity from Spanish germplasm, including the sweet cultivar Fuentes de Ebro, the highly pungent landrace BGHZ1354 and the two Valenciana type commercial varieties Recas and Rita, was evaluated and optimized in this study. The OH-1 population, characterized by a high gynogenesis induction, was used as control. Growing conditions of the donor plants were tested with a one-step protocol and field plants produced a slightly higher percentage of embryogenesis induction than growth chamber plants. A one-step protocol was compared with a two-step protocol for embryogenesis induction. Spanish germplasm produced a 23 times higher percentage of embryogenesis with the two-step protocol, Recas showing the highest percentage (2.09%) and Fuentes de Ebro the lowest (0.53%). These percentages were significantly lower than those from the OH-1 population, with an average of 15% independently of the protocol used. The effect of different containers on plant regeneration was tested using both protocols. The highest percentage of acclimated plants was obtained with the two-step protocol in combination with Eco2box (70%), whereas the lowest percentage was observed with glass tubes in the two protocols (2023%). Different amiprofos-methyl (APM) treatments were applied to embryos for chromosome doubling. A similar number of doubled haploid plants were recovered with 25 or 50 ?M APM in liquid medium. However, the application of 25 ?M in solid medium for 24 h produced the highest number of doubled haploid plants. Somatic regeneration from flower buds of haploid and mixoploid plants proved to be a successful approach for chromosome doubling, since diploid plants were obtained from the four regenerated lines. In this study, doubled haploid plants were produced from the four Spanish cultivars, however further improvements are needed to increase their gynogenesis efficiency.

The effects of acrolein were studied on the chick embryos of 48 and 72 hr of incubation. Acrolein was dissolved in physiological saline and injected into the air sacs of the eggs at doses ranging from 0.001 to 0.1 mg per egg. The controls received and equal amount of saline only (0.1 ml per egg). All the embryos including controls were examined at Day 13. In all, 600 eggs were utilized for this investigation. At 48 hr incubation, the percentage survival ranged from 80 to 0 as the dosage of acrolein was increased. Embryonic mortality following 72 hr incubation did not increase significantly at any dose level. Gross malformations such as short and twisted limbs, everted viscera, microphthalmia, short and twisted neck, and hemorrhage over the body were observed. The frequency and the types of gross abnormalities did not vary much in the 48- or 72-hr-treated groups. The incidence of malformation in the controls was low. The results of this study indicates that acrolein is embryotoxic at higher doses and moderately teratogenic to chick embryogenesis.

Temperature affects both the timing and outcome of animal development, but the detailed effects of temperature on the progress of early development have been poorly characterized. To determine the impact of temperature on the order and timing of events during Drosophila melanogaster embryogenesis, we used time-lapse imaging to track the progress of embryos from shortly after egg laying through hatching at seven precisely maintained temperatures between 17.5°C and 32.5°C. We employed a combination of automated and manual annotation to determine when 36 milestones occurred in each embryo. D. melanogaster embryogenesis takes 33 hours at 17.5°C, and accelerates with increasing temperature to a low of 16 hours at 27.5°C, above which embryogenesis slows slightly. Remarkably, while the total time of embryogenesis varies over two fold, the relative timing of events from cellularization through hatching is constant across temperatures. To further explore the relationship between temperature and embryogenesis, we expanded our analysis to cover ten additional Drosophila species of varying climatic origins. Six of these species, like D. melanogaster, are of tropical origin, and embryogenesis time at different temperatures was similar for them all. D. mojavensis, a sub-tropical fly, develops slower than the tropical species at lower temperatures, while D. virilis, a temperate fly, exhibits slower development at all temperatures. The alpine sister species D. persimilis and D. pseudoobscura develop as rapidly as tropical flies at cooler temperatures, but exhibit diminished acceleration above 22.5°C and have drastically slowed development by 30°C. Despite ranging from 13 hours for D. erecta at 30°C to 46 hours for D. virilis at 17.5°C, the relative timing of events from cellularization through hatching is constant across all species and temperatures examined here, suggesting the existence of a previously unrecognized timer controlling the progress of embryogenesis that has been tuned by natural selection as each species diverges. PMID:24762628

Background The standard textbook information that annelid musculature consists of oligochaete-like outer circular and inner longitudinal muscle-layers has recently been called into question by observations of a variety of complex muscle systems in numerous polychaete taxa. To clarify the ancestral muscle arrangement in this taxon, we compared myogenetic patterns during embryogenesis of Ophryotrocha diadema with available data on oligochaete and polychaete myogenesis. This work addresses the conflicting views on the ground pattern of annelids, and adds to our knowledge of the evolution of lophotrochozoan taxa. Results Somatic musculature in Ophryotrocha diadema can be classified into the trunk, prostomial/peristomial, and parapodial muscle complexes. The trunk muscles comprise strong bilateral pairs of distinct dorsal and ventral longitudinal strands. The latter are the first to differentiate during myogenesis. They originate within the peristomium and grow posteriorly through the continuous addition of myocytes. Later, the longitudinal muscles also expand anteriorly and form a complex arrangement of prostomial muscles. Four embryonic parapodia differentiate in an anterior-to-posterior progression, significantly contributing to the somatic musculature. Several diagonal and transverse muscles are present dorsally. Some of the latter are situated external to the longitudinal muscles, which implies they are homologous to the circular muscles of oligochaetes. These circular fibers are only weakly developed, and do not appear to form complete muscle circles. Conclusion Comparison of embryonic muscle patterns showed distinct similarities between myogenetic processes in Ophryotrocha diadema and those of oligochaete species, which allows us to relate the diverse adult muscle arrangements of these annelid taxa to each other. These findings provide significant clues for the interpretation of evolutionary changes in annelid musculature. PMID:18171469

In 2006, Dr Shinya Yamanaka succeeded to reprogram somatic cells into pluripotent stem cells (iPSC) by delivering the genes encoding Oct4, Sox2, Klf4, and c-Myc. This achievement represents a fundamental breakthrough in stem cell biology and opens up a new era in regenerative medicine. However, the molecular processes by which somatic cells are reprogrammed into iPSC remain poorly understood. In 2009, Yamanaka proposed the elite and stochastic models for reprogramming mechanisms. To date, many investigators in the field of iPSC research support the concept of stochastic model, i.e., somatic cell reprogramming is an event of epigenetic transformation. A mathematical model, f (Cd, k), has also been proposed to predict the stochastic process. Here we wish to revisit the Yamanaka model and summarize the recent advances in this research field. PMID:24189530

An efficient in vitro protocol has been established for somaticembryogenesis and plantlet conversion of Korean wild ginseng (Panax ginseng Meyer). Wild-type and mutant adventitious roots derived from the ginseng produced calluses on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid and 0.3 mg/L kinetin; 53.3% of the explants formed callus. Embryogenic callus proliferation and somatic embryo induction occurred on MS medium containing 0.5 mg/L 2,4-dichlorophenoxyacetic acid. The inducedsomatic embryos further developed to maturity on MS medium with 5 mg/L gibberellic acid, and 85% of them germinated. The germinated embryos were developed to shoots and elongated on MS medium with 5 mg/L gibberellic acid. The shoots developed into plants with well-developed taproots on one-third strength Schenk and Hildebrandt basal medium supplemented with 0.25 mg/L 1-naphthaleneacetic acid. When the plants were transferred to soil, about 30% of the regenerated plants developed into normal plants. PMID:25378998

Receptor-like kinases are important regulators of plant growth. Often a single receptor is involved in regulation of multiple developmental processes in a variety of tissues. ERECTA family (ERf) receptors have previously been linked with stomata development, above-ground organ elongation, shoot apical meristem function, flower differentiation and biotic/abiotic stresses. Here we explore the role of these genes during embryogenesis. ERfs are expressed in the developing embryo, where their expression is progressively limited to the upper half of the embryo. During embryogenesis ERfs redundantly stimulate the growth of cotyledons by promoting cell proliferation and inhibiting premature stomata differentiation. PMID:25240196

In general, it had been believed that the cell fate restriction of terminally differentiated somatic cells was irreversible. In 1952, somatic cell nuclear transfer (SCNT) was introduced to study early embryonic development in frogs. So far, various mammalian species have been successfully cloned using the SCNT technique, though its efficiency is very low. Embryonic stem (ES) cells were the first pluripotent cells to be isolated from an embryo and have a powerful potential to differentiate into more than 260 types of cells. The generation of induced pluripotent stem (iPS) cells was a breakthrough in stem cell research, and the use of these iPS cells has solved problems such as low efficiency and cell fate restriction. These cells have since been used for clinical application, disease investigation, and drug selection. As it is widely accepted that the endosymbiosis of Archaea into eukaryotic ancestors resulted in the generation of eukaryotic cells, we examined whether bacterial infection could alter host cell fate. We previously showed that when human dermal fibroblast (HDF) cells were incorporated with lactic acid bacteria (LAB), the LAB-incorporated HDF cells formed clusters and expressed a subset of common pluripotent markers. Moreover, LAB-incorporated cell clusters could differentiate into cells derived from each of the three germinal layers both in vivo and in vitro, indicating successful reprogramming of host HDF cells by LAB. In the current review, we introduce the existing examples of cellular reprogramming by bacteria and discuss their nuclear reprogramming mechanisms. PMID:25866152

Somatic treatments for mood disorders represent a class of interventions available either as a stand-alone option, or in combination with psychopharmacology and\\/or psychotherapy. Here, we review the currently available techniques, including those already in clinical use and those still under research. Techniques are grouped into the following categories: (1) seizure therapies, including electroconvulsive therapy and magnetic seizure therapy, (2) noninvasive

An investigation of somatic variation in 10 plants of Lolium perenne, using a two-stage cloning process followed by two further cycles of vegetative propagation, has revealed that persistent differences in tiller number and plant height may arise at the time of the initial cloning. These effects were dependent upon the age of the clone and its past vegetative history. Transmissibility

INTRODUCTION During early Drosophila embryogenesis, a number of developmental programs unfold and Nüsslein-Vol- hard, 1988; reviewed by Govind and Steward, 1991). Later, during germ band extension the ectoderm give rise to about 250 neurons. During germ band retraction, the CNS continues to differentiate

(reviewed by Grinnell, 1992; Martin, 1997; Werner and Grose, 2003). The deeper connective tissue is replaced of collagen within the healed connective tissue. Tissue repair in the mouse embryo involves largely the same3021 Introduction Building tissues and organs during embryogenesis involves a series of exquisite

Early events leading to fate decisions during leech embryogenesis Marc Pilon and David A. Weisblat This paper reviews leech development up to the 12-cell embryo. Oogenesis proceeds by a system of nurse cells-nos, a leech homolog to the Drosophila gene nanos, suggests that it may be a determinant associated

Genetic mutations causing human disease are conventionally thought to be inherited through the germ line from ones parents and present in all somatic (body) cells, except for most cancer mutations, which arise somatically. Increasingly, somatic mutations are being identified in diseases other than cancer, including neurodevelopmental diseases. Somatic mutations can arise during the course of prenatal brain development and cause neurological diseaseeven when present at low levels of mosaicism, for exampleresulting in brain malformations associated with epilepsy and intellectual disability. Novel, highly sensitive technologies will allow more accurate evaluation of somatic mutations in neurodevelopmental disorders and during normal brain development. PMID:23828942

We analyzed DNA sequences that regulate the expression of an isocitrate lyase gene from Brassica napus L. during late embryogenesis and during postgerminative growth to determine whether glyoxysomal function is induced by a common mechanism at different developmental stages. beta-Glucuronidase constructs were used both in transient expression assays in B. napus and in transgenic Arabidopsis thaliana to identify the segments of the isocitrate lyase 5' flanking region that influence promoter activity. DNA sequences that play the principal role in activating the promoter during post-germinative growth are located more than 1,200 bp upstream of the gene. Distinct DNA sequences that were sufficient for high-level expression during late embryogenesis but only low-level expression during postgerminative growth were also identified. Other parts of the 5' flanking region increased promoter activity both in developing seed and in seedlings. We conclude that a combination of elements is involved in regulating the isocitrate lyase gene and that distinct DNA sequences play primary roles in activating the gene in embryos and in seedlings. These findings suggest that different signals contribute to the induction of glyoxysomal function during these two developmental stages. We also showed that some of the constructs were expressed differently in transient expression assays and in transgenic plants. PMID:8934622

After pre-culture and treatment of osmosis, zygotic embryos of peanut (Arachis hypogaea L.) were transformed via particle bombardment with a plasmid containing a Bluetongue VP2 gene (BTVP2) comprising neutralizing epitopes. Selection for Kanamycin resistant calluses and somatic embryos was initiated at 12th day post-bombardment on medium containing 25 mg/L Kanamycin. Under continuous selection, 12.38% Kanamycin resistant plantlets were regenerated from bombarded somatic embryos. The presence and integration of BTVP2 DNA in regenerated Kanamycin resistant plants were confirmed by southern hybridization assay using non-radioactive Digoxiginin BTVP2 probe. Beta-glucuronidase (GUS) enzyme activity was detected in transgenic somatic embryos but not from control, non-transformed embryos. The expression of the BTVP2 protein was confirmed through RT-PCR (reverse transcription polymerase chain reaction) using the RNA isolated from the transgenic callus employing BTVP2-specific primers. The production of transgenic peanut was mainly focused on evaluating a newly improved somaticembryogenesis regeneration system as well as the gene transfer method and to produce the Bluetongue outer coat protein that comprises the neutralizing epitopes. PMID:16513222

Cotyledonary somatic embryos of white spruce [Picea glauca (Moench) Voss] were subjected to microprojectile bombardment with a gene construct containing a gus::nptll fusion gene. Somatic embryos were used to re-induce the embryogenic tissue after bombardments. Histochemical assay using X-gluc as a substrate showed that all the embryos (100%) were GUS positive 48 h after bombardment. However, only thirteen out of

Directed reprogramming of somatic cells by defined factors provides a novel method for the generation of patient-specific stem cells with the potential to bypass both the practical and ethical concerns associated with somatic cell nuclear transfer (SCNT) and human embryonic stem (hES) cells. Although the generation of induced pluripotent stem (iPS) cells has proven a robust technology in mouse and

Between February and December, 1977, 133,493 test-day observations of somatic cell count were taken on 27,009 Holstein cows in 676 herds on the Quebec Dairy Herd Analysis Service. Data were transformed to a log (natural) scale, and analyses were separate within lacta- tion age group (42, 3, 4, 5, and \\/>6 yr). Joint estimates of fixed effects of month of

Adult attachment representations have been considered to play a role in the development and treatment of somatizing behavior. In this study, the associations between the two attachment dimensions avoidance and anxiety and dimensions of psychopathology (somatization, depression, and general anxiety) were explored. The sample consists of 202 outpatients diagnosed with a somatoform disorder. Data were collected via self-report measures. A path analysis shows that the two attachment dimensions are not directly associated with somatization. There are, however, significant indirect associations between attachment and somatization mediated by depression and general anxiety, which are more pronounced for attachment anxiety than for attachment avoidance. The findings reveal that a low level of attachment security in romantic relationships, especially an anxious stance toward the partner, comes along with poor mental health, which in turn is related to a preoccupation with somatic complaints. Implications for the treatment of somatizing patients are discussed. PMID:25594785

The fragmenting potworm Enchytraeus japonensis (Oligochaeta, Annelida) reproduces asexually by dividing the body into several fragments that then regenerate to complete individuals in 4-5 days. Such large-scale regeneration, however, occurs only in some invertebrates. To better our understanding of why regeneration is so limited in many animals, despite their ability to undergo embryonic development from the single cell of a fertilized egg, comparisons were made between regeneration and embryonic development of E. japonensis by using two methods: histochemistry for alkaline phosphatase (ALP) and immunohistochemistry with an antibody against acetylated tubulin that visualizes nervous system development. The analyses revealed that both ALP expression patterns and central nervous system development differ between embryogenesis and the regeneration, suggesting that regeneration is not a simple reiteration of embryogenesis but involves different regulatory mechanisms. The study provides a basis for the elucidation of mechanisms that are unique and crucial to regeneration. PMID:15366012

All multicellular living beings are created from a single cell. A developmental process, called embryogenesis, takes this first fertilized cell down a complex path of reproduction, migration, and specialization into a complex organism adapted to its environment. In most cases, the first steps of the embryogenesis take place in a protected environment such as in an egg or in utero. Starting from this observation, we propose a new approach to the generation of real robots, strongly inspired by living systems. Our robots are composed of tens of specialized cells, grown from a single cell using a bio-inspired virtual developmental process. Virtual cells, controlled by gene regulatory networks, divide, migrate, and specialize to produce the robot's body plan (morphology), and then the robot is manually built from this plan. Because the robot is as easy to assemble as Lego, the building process could be easily automated. PMID:24730763

The paper presents selected figures of chick embryogenesis as depicted in the classic studies of Caspar Friedrich Wolff (17341794), Christian Heinrich Pander (17941865) and Karl Ernst von Baer (17921786). My main objective here is (1) to demonstrate how the imagery of Wolff, Pander and Baer attempted to project an image of a 3-dimensional rotating body into static figures on paper

\\u000a During fertilization, the sperm delivers a haploid set of chromosomes to the zygote. Genetic alterations, such as numerical\\u000a or structural chromosome defects, can affect the ability of the embryo to undergo normal development. Similarly, epigenetic\\u000a defects, such as abnormal methylation of gene promoters, may affect gene expression during embryogenesis and affect the viability\\u000a or health of the developing embryo. This

?-Tocopherol is a lipid-soluble antioxidant that is specifically required for reproduction and embryogenesis. However, since its discovery, ?-tocopherol's specific biologic functions, other than as an antioxidant, and the mechanism(s) mediating its requirement for embryogenesis remain unknown. As an antioxidant, ?-tocopherol protects polyunsaturated fatty acids (PUFAs) from lipid peroxidation. ?-Tocopherol is probably required during embryonic development to protect PUFAs that are crucial to development, specifically arachidonic (ARA) and docosahexaenoic (DHA) acids. Additionally, ARA and DHA are metabolized to bioactive lipid mediators via lipoxygenase enzymes, and ?-tocopherol may directly protect, or it may mediate the production and/or actions of, these lipid mediators. In this review, we discuss how ?-tocopherol (1) prevents the nonspecific, radical-mediated peroxidation of PUFAs, (2) functions within a greater antioxidant network to modulate the production and/or function of lipid mediators derived from 12- and 12/15-lipoxygenases, and (3) modulates 5-lipoxygenase activity. The application and implication of such interactions are discussed in the context of ?-tocopherol requirements during embryogenesis. PMID:23920314

The present study aimed at developing temporary immersion bioreactor techniques for multiplication of cacao somatic embryos. Temporary Immersion System (TIS), i.e. flooding of plant tissue at regular time intervals provides an efficient way to propagate plants. Somatic embryos were regenerated in twin flask bioreactors. The TIS proved to be suitable for mass regeneration of somatic embryos and for their subsequent direct sowing. The number of embryos after 3 months of culture was significantly higher in TIS cultures than in the solid medium variant. TIS also improved embryo development regarding the conversion to torpedo shaped forms. Matured embryos derived from TIS and pre-treated with 6% sucrose were converted into plants after direct sowing. Additionally to the influence of culture conditions on the development of somaticembryogenesis the content and composition of free amino acids were analysed. The content of free amino acids in somatic embryos rose as immersion frequency increased. The endogenous free GABA content in embryogenic callus was significantly higher than in non-embryogenic callus. PMID:18193427

Earlier studies found that cotton (Gossypium hirsutum L.) cotyledons contain several mRNAs which are more abundant during late embryogenesis than in mid-embryogenesis or early germination. They are here termed Late embryogenesis-abundant mRNAs, encoded by Lea loci. Complementary DNA clones for 18 such mRNA sequences, defined at a hybridization criterion of Tm-15°C, were identified in a mature embryo cDNA library by

Studies in vertebrate and invertebrate model organisms on the molecular basis of primordial germ cell (PGC) specification have revealed that metazoans can specify their germ line either early in development by maternally transmitted cytoplasmic factors (inheritance), or later in development by signaling factors from neighboring tissues (induction). Regardless of the mode of PGC specification, once animal germ cells are specified, they invariably express a number of highly conserved genes. These include vasa and piwi, which can play essential roles in any or all of PGC specification, development, or gametogenesis. Although the arthropods are the most speciose animal phylum, to date there have been no functional studies of conserved germ line genes in species of the most basally branching arthropod clade, the chelicerates (which includes spiders, scorpions, and horseshoe crabs). Here we present the first such study by using molecular and functional tools to examine germ line development and the roles of vasa and piwi orthologues in the common house spider Parasteatoda (formerly Achaearanea) tepidariorum. We use transcript and protein expression patterns of Pt-vasa and Pt-piwi to show that primordial germ cells (PGCs) in the spider arise during late embryogenesis. Neither Pt-vasa nor Pt-piwi gene products are localized asymmetrically to any embryonic region before PGCs emerge as paired segmental clusters in opisthosomal segments 2-6 at late germ band stages. RNA interference studies reveal that both genes are required maternally for egg laying, mitotic progression in early embryos, and embryonic survival. Our results add to the growing body of evidence that vasa and piwi can play important roles in somatic development, and provide evidence for a previously hypothesized conserved role for vasa in cell cycle progression. PMID:25257304

Cell fusion is a potent approach to explore the mechanisms of somatic cells reprogramming. However, previous fusion methods, such as polyethylene glycol (PEG) mediated cell fusion, are often limited by poor fusion yields. In this study, we developed a simplified cell electrofusion chip, which was based on a micro-cavity/ discrete microelectrode structure to improve the fusion efficiency and to reduce multi-cell electrofusion. Using this chip, we could efficiently fuse NIH3T3 cells and mouse embryonic stem cells (mESCs) to inducesomatic cells reprogramming. We also found that fused cells demethylated gradually and 5-hydroxymethylcytosine (5hmC) was involved in the demethylation during the reprogramming. Thus, the cell electrofusion chip would facilitate reprogramming mechanisms research by improving efficiency of cell fusion and reducing workloads. PMID:26177036

The purpose of this study was to evaluate and compare the use of liquid and solid Murashige and Skoog (MS) medium in different culture vessels for mass production of Catharanthus roseus, an important source of anticancerous compounds, vincristine and vinblastine. Three media conditions i.e. agar-solidified medium (S), liquid medium in agitated conical flask (L) and growtek bioreactor (B) were used. Rapid propagation was achieved through in vitro somaticembryogenesis pathway. The process of embryogenesis has been categorized into induction, proliferation, maturation and germination stages. All in vitro embryogenesis stages were conducted by withdrawing spent liquid medium and by adding fresh MS medium. In optimized 4.52 ?M 2,4-D added MS, the callus biomass growth was low in solid (1.65 g) compared to liquid medium in agitated conical flask (1.95 g) and in bioreactor (2.11 g). The number of normal somatic embryos was more in solid medium (99.75/50 mg of callus mass) compared to liquid medium used in conical flask (83.25/callus mass) and growtek bioreactor (84.88/callus mass). The in vitro raised embryos maturated in GA3 (2.60 ?M) added medium; and in bioreactor the embryo growth was high, a maximum length of 9.82 mm was observed at the end of four weeks. These embryos germinated into seedlings in BAP (2.22 ?M) added medium and the embryo germination ability was more (59.41%) in bioreactor compared to liquid medium in conical flask (55.5%). Shoot length (11.25 mm) was also high in bioreactor compared to agitated conical flask. The liquid medium used in agitated conical flask and bioreactor increased seedling production efficiency, at the same time it also reduced plant recovery time. The embryo generated plants grew normally in outdoor conditions. The exploitation of medium to large culture vessel or bioreactor may make the process more efficient in getting large number of Catharanthus plant as it is the only source of anti-cancerous alkaloids, vincristine and vinblastine. PMID:25313279

The purpose of this study was to evaluate and compare the use of liquid and solid Murashige and Skoog (MS) medium in different culture vessels for mass production of Catharanthus roseus, an important source of anticancerous compounds, vincristine and vinblastine. Three media conditions i.e. agar-solidified medium (S), liquid medium in agitated conical flask (L) and growtek bioreactor (B) were used. Rapid propagation was achieved through in vitro somaticembryogenesis pathway. The process of embryogenesis has been categorized into induction, proliferation, maturation and germination stages. All in vitro embryogenesis stages were conducted by withdrawing spent liquid medium and by adding fresh MS medium. In optimized 4.52 ?M 2,4-D added MS, the callus biomass growth was low in solid (1.65 g) compared to liquid medium in agitated conical flask (1.95 g) and in bioreactor (2.11 g). The number of normal somatic embryos was more in solid medium (99.75/50 mg of callus mass) compared to liquid medium used in conical flask (83.25/callus mass) and growtek bioreactor (84.88/callus mass). The in vitro raised embryos maturated in GA3 (2.60 ?M) added medium; and in bioreactor the embryo growth was high, a maximum length of 9.82 mm was observed at the end of four weeks. These embryos germinated into seedlings in BAP (2.22 ?M) added medium and the embryo germination ability was more (59.41%) in bioreactor compared to liquid medium in conical flask (55.5%). Shoot length (11.25 mm) was also high in bioreactor compared to agitated conical flask. The liquid medium used in agitated conical flask and bioreactor increased seedling production efficiency, at the same time it also reduced plant recovery time. The embryo generated plants grew normally in outdoor conditions. The exploitation of medium to large culture vessel or bioreactor may make the process more efficient in getting large number of Catharanthus plant as it is the only source of anti-cancerous alkaloids, vincristine and vinblastine. PMID:25313279

We describe a simple and efficient protocol for regeneration-transformation of two diploid Medicago lines: the annual M. truncatula R108-1(c3) and the perennial M. sativa ssp. falcata (L.) Arcangeli PI.564263 selected previously as highly embryogenic genotypes. Here, embryo regeneration of R108-1 to complete\\u000a plants was further improved by three successive in vitro regeneration cycles resulting in the line R108-1(c3). Agrobacterium tumefaciens-mediated

Summary The frequency and quality of embryogenic response from cotyledons of immature zygotic soybean embryos varied with 2,4-dichlorophenoxyacetic\\u000a acid (2,4-D) concentration in the culture medium. The frequency of variants among progeny of regenerated plants decreased\\u000a with an increase of 2,4-D concentration. Teratogenic effects on embryo morphology and development were greatest at 22.5µM 2,4-D and decreased with increasing 2,4-D. At the lowest

Callus culture was initiated from expiants of mature root tissues of ginseng (Panax ginseng C.A. Meyer) on MS medium enriched with 2,4-D. The ageing callus produced numerous embryoids in this medium. Reculture of these embryoids in media (1\\/2 MS or B5) supplemented with benzyladenine and gibberellic acid resulted in profuse plantlet regeneration.

Callus growth has been observed from plumules of ecotype Laguna Tall after cryopreservation using an encapsulation/dehydration protocol. Sucrose preculture treatment and silica gel dehydration both significantly influenced the frequency of callus formation from non-frozen and frozen plumules. The greatest frequency of post-thaw callus growth occurred after incubation of the encapsulated plumules for 72-96 h in medium containing 0.75 M sucrose followed by desiccation over silica gel for 7-8 h down to approximately 30% moisture content (fresh weight basis). Freezing and thawing were carried out rapidly. Post-thaw recovery rates in excess of 80% were recorded. PMID:11788861

The scarcity and stochastic nature of genetic mutations presents a significant challenge for scientists seeking to characterise\\u000a de novo mutation frequency at specific loci. Such mutations can be particularly numerous during regeneration of plants from\\u000a in vitro culture and can undermine the value of germplasm conservation efforts. We used cleaved amplified polymorphic sequence\\u000a (CAPS) analysis to characterise new mutations amongst

In flowering plants, male gametophytes are generated in anthers from microsporocytes. However, more evidence is needed to reveal the genetic mechanisms which regulate the differentiation and interaction of these highly specialized cells in anthers. Here we report the characterization of a series of male-sterile cotton (Gossypium hirsutum) mutants, including mutants with normal fertility, semi-sterility and complete sterility. These mutants are forms of transgenic cotton containing RNAi vectors with partial cDNA fragments of GhSERK1. The GhSERK1 gene encodes a putative leucine-rich repeat receptor protein kinase (LRR-RLK), and generally has 11 domains. In previous research, we found plants containing GhSERK1 produce an abundance of male reproductive tissue. In this paper, three RNAi constructs were designed separately to analyze its function in anther. After the three RNAi vectors were transformed into the cotton, transgenic plants with the specialized fragment exhibited normal fertility or the pollen energy decreased slightly, as ones with the homologous fragments exhibited various degrees of male sterility with different expression levels of GhSERK1 mRNA. In conclusion, for the transgenic plants with conserved fragments, lower expression levels of GhSERK1 mRNA were in transgenic plants, and a higher degree of male sterility was observed. Taking together, these findings demonstrate the GhSERK1 gene has a role in the development of anthers, especially in the formation of pollen grains. Also, we infer there must be another homolog of GhSERK1 in cotton, and both of GhSERK1 and its homolog function redundantly as important control points in controlling anther pollen production. PMID:24276918

Protoplasts were isolated from cell suspensions derived from cotyledon and hypocotyl Gentiana kurroo (Royle). Cell walls were digested with an enzyme cocktail containing cellulase, macerozyme, driselase, hemicellulase and\\u000a pectolyase in CPW solution. Protoplast viability ranged from 88 to 96%. Three techniques of culture and six media were evaluated\\u000a in terms of their efficiency in producing viable cultures and regenerating whole

Embryogenic callus from four asparagus genotypes, Jersey Giant No. 8, MD10, Rutgers 22, and 86SOM1 was simultaneously initiated from spear explants on semisolid LS medium containing 5 ?M 2,4-D or 50 ?M NAA. Calluses were used to initiate cell suspensions in liquid LS medium of the same composition. The eight sets of cell suspensions were used as protoplast donors at

The authors review the research on childhood antecedents and personality contributions to the somatoform disorders, as well as research on social influences during adulthood. Based on these data, the authors hypothesize that somatizing patients display anxious attachment behavior that derives from childhood experiences with caregivers. Early exposure to illness increases the likeli- hood that distress will be manifested somatically. When

Somatic mosaicism refers to the occurrence of two genetically distinct populations of cells within an individual, derived from a postzygotic mutation. In contrast to inherited mutations, somatic mosaic mutations may affect only a portion of the body and are not transmitted to progeny. These mutations affect varying genomic sizes ranging from single nucleotides to entire chromosomes and have been implicated in disease, most prominently cancer. The phenotypic consequences of somatic mosaicism are dependent upon many factors including the developmental time at which the mutation occurs, the areas of the body that are affected, and the pathophysiological effect(s) of the mutation. The advent of second-generation sequencing technologies has augmented existing array-based and cytogenetic approaches for the identification of somatic mutations. We outline the strengths and weaknesses of these techniques and highlight recent insights into the role of somatic mosaicism in causing cancer, neurodegenerative, monogenic, and complex disease. PMID:25513881

The zebrafish Danio rerio has emerged as a powerful vertebrate model system that lends itself particularly well to quantitative investigations with live imaging approaches, owing to its exceptionally high optical clarity in embryonic and larval stages. Recent advances in light microscopy technology enable comprehensive analyses of cellular dynamics during zebrafish embryonic development, systematic mapping of gene expression dynamics, quantitative reconstruction of mutant phenotypes and the system-level biophysical study of morphogenesis. Despite these technical breakthroughs, it remains challenging to design and implement experiments for in vivo long-term imaging at high spatio-temporal resolution. This article discusses the fundamental challenges in zebrafish long-term live imaging, provides experimental protocols and highlights key prop1erties and capabilities of advanced fluorescence microscopes. The article focuses in particular on experimental assays based on light sheet-based fluorescence microscopy, an emerging imaging technology that achieves exceptionally high imaging speeds and excellent signal-to-noise ratios, while minimizing light-induced damage to the specimen. This unique combination of capabilities makes light sheet microscopy an indispensable tool for the in vivo long-term imaging of large developing organisms. PMID:23523701

This report describes the regeneration of plants from callus cultures of caraway, Carum carvi L. Callus of hypocotyl origin was maintained on medium containing Murashige and Skoog salts with Nitsch and Nitsch vitamins in the presence of 0.3 mg · l(-1) 2,4-D. Formation of somatic embryos was induced with suspension cultures once 2,4-D had been removed. Embryos developed into plantlets when subcultured on solid medium supplemented with 0.5 mg · l(-1) IBA and 10.0 mg · l(-1) adenine sulphate. Regenerated plantlets were transferred to soil. PMID:23194576

Long noncoding RNAs (lncRNAs) comprise a diverse class of transcripts that structurally resemble mRNAs but do not encode proteins. Recent genome-wide studies in humans and the mouse have annotated lncRNAs expressed in cell lines and adult tissues, but a systematic analysis of lncRNAs expressed during vertebrate embryogenesis has been elusive. To identify lncRNAs with potential functions in vertebrate embryogenesis, we performed a time-series of RNA-seq experiments at eight stages during early zebrafish development. We reconstructed 56,535 high-confidence transcripts in 28,912 loci, recovering the vast majority of expressed RefSeq transcripts while identifying thousands of novel isoforms and expressed loci. We defined a stringent set of 1133 noncoding multi-exonic transcripts expressed during embryogenesis. These include long intergenic ncRNAs (lincRNAs), intronic overlapping lncRNAs, exonic antisense overlapping lncRNAs, and precursors for small RNAs (sRNAs). Zebrafish lncRNAs share many of the characteristics of their mammalian counterparts: relatively short length, low exon number, low expression, and conservation levels comparable to that of introns. Subsets of lncRNAs carry chromatin signatures characteristic of genes with developmental functions. The temporal expression profile of lncRNAs revealed two novel properties: lncRNAs are expressed in narrower time windows than are protein-coding genes and are specifically enriched in early-stage embryos. In addition, several lncRNAs show tissue-specific expression and distinct subcellular localization patterns. Integrative computational analyses associated individual lncRNAs with specific pathways and functions, ranging from cell cycle regulation to morphogenesis. Our study provides the first systematic identification of lncRNAs in a vertebrate embryo and forms the foundation for future genetic, genomic, and evolutionary studies. PMID:22110045

Ionizing radiation (IR) treatment induces a DNA damage response, including cell cycle arrest, DNA repair, and apoptosis in metazoan somatic cells. Because little has been reported in germline cells, we performed a temporal analysis of the DNA damage response utilizing Drosophila oogenesis as a model system. Oogenesis in the adult Drosophila female begins with the generation of 16-cell cyst by four mitotic divisions of a cystoblast derived from the germline stem cells. We found that high-dose irradiation induced S and G2 arrests in these mitotically dividing germline cells in a grp/Chk1- and mnk/Chk2-dependent manner. However, the upstream kinase mei-41, Drosophila ATR ortholog, was required for the S-phase checkpoint but not for the G2 arrest. As in somatic cells, mnk/Chk2 and dp53 were required for the major cell death observed in early oogenesis when oocyte selection and meiotic recombination occurs. Similar to the unscheduled DNA double-strand breaks (DSBs) generated from defective repair during meiotic recombination, IR-induced DSBs produced developmental defects affecting the spherical morphology of meiotic chromosomes and dorsal-ventral patterning. Moreover, various morphological abnormalities in the ovary were detected after irradiation. Most of the IR-induced defects observed in oogenesis were reversible and were restored between 24 and 96 h after irradiation. These defects in oogenesis severely reduced daily egg production and the hatch rate of the embryos of irradiated female. In summary, irradiated germline cells induced DSBs, cell cycle arrest, apoptosis, and developmental defects resulting in reduction of egg production and defective embryogenesis. PMID:24551207

LETTERS Cytokinin and auxin interaction in root stem-cell specification during early embryogenesisembryogenesis2,3 , but the early embryonic function of cytokinin is obscure4­6 . Here, we introduce a synthetic reporter to visualize universally cytokinin output in vivo. Notably, the first embryonic signal is detected

Pluripotent stem cells, which are capable of differentiating in various species of cells, are hoped to be donor cells in transplantation in regenerative medicine. Embryonic stem (ES) cells and induced pluripotent stem cells have the potential to differentiate in approximately all species of cells. However, the proliferating ability of these cells is high and the cancer formation ability is also recognized. In addition, ethical problems exist in using ES cells. Somatic stem cells with the ability to differentiate in various species of cells have been used as donor cells for neuronal diseases, such as amyotrophic lateral sclerosis, spinal cord injury, Alzheimer disease, cerebral infarction and congenital neuronal diseases. Human mesenchymal stem cells derived from bone marrow, adipose tissue, dermal tissue, umbilical cord blood and placenta are usually used for intractable neuronal diseases as somatic stem cells, while neural progenitor/stem cells and retinal progenitor/stem cells are used for a few congenital neuronal diseases and retinal degenerative disease, respectively. However, non-treated somatic stem cells seldom differentiate to neural cells in recipient neural tissue. Therefore, the contribution to neuronal regeneration using non-treated somatic stem cells has been poor and various differential trials, such as the addition of neurotrophic factors, gene transfer, peptide transfer for neuronal differentiation of somatic stem cells, have been performed. Here, the recent progress of regenerative therapies using various somatic stem cells is described. PMID:24179604

Highlights: The Gper expression was detected in the developing brain of zebrafish. Gper morpholino knockdown induced apoptosis of brain cells. Gper morpholino knockdown reduced expression in neuron markers. Zebrafish Gper may be involved in neuronal development. -- Abstract: G-protein-coupled estrogen receptor 1 (Gper, formerly known as GPR30) is found to be a trophic and protective factor in mediating action of estrogen in adult brain, while its role in developing brain remains to be elucidated. Here we present the expression pattern of Gper and its functions during embryogenesis in zebrafish. Both the mRNA and protein of Gper were detected throughout embryogenesis. Whole mount in situ hybridization (WISH) revealed a wide distribution of gper mRNAs in various regions of the developing brain. Gper knockdown by specific morpholinos resulted in growth retardation in embryos and morphological defects in the developing brain. In addition, induced apoptosis, decreased proliferation of the brain cells and maldevelopment of sensory and motor neurons were also found in the morphants. Our results provide novel insights into Gper functions in the developing brain, revealing that Gper can maintain the survival of the brain cells, and formation and/or differentiation of the sensory and motor neurons.

The process of somatic hypermutation (SHM) of immunoglobulin (Ig) genes requires activation-induced cytidine deaminase (AID). Although mistargeting of AID is detrimental to genome integrity, the mechanism and the cis-elements responsible for targeting of AID are largely unknown. We show that three CAGGTG cis-elements in the context of Ig enhancers are sufficient to target SHM to a nearby transcribed gene. The CAGGTG motif binds E47 in nuclear extracts of the mutating cells. Replacing CAGGTG with AAGGTG in the construct without any other E47 binding site eliminates SHM. The CA versus AA effect requires AID. CAGGTG does not enhance transcription, chromatin acetylation, or overall target gene activity. The other cis-elements of Ig enhancers alone cannot attract the SHM machinery. Collectively with other recent findings, we postulate that AID targets all genes expressed in mutating B cells that are associated with CAGGTG motifs in the appropriate context. Ig genes are the most highly mutated genes, presumably because of multiple CAGGTG motifs within the Ig genes, high transcription activity, and the presence of other cooperating elements in Ig enhancers. PMID:20100870

In this article the use of somatic cell counts for monitoring udder health and milk quality is discussed. Somatic cell count dynamics at quarter, cow, herd and population level are discussed and illustrated with examples. Quarter and cow somatic cell counts directly represent the inflammatory status of the mammary gland. Herd and population somatic cell count are related to the

Somatic symptoms are an associated feature of anxiety disorders that have received little research attention among non-White samples. In addition, the majority of previous studies have examined the influence of somatic symptoms in a cross-sectional rather than a prospective manner. This study examines the prevalence of 12 somatic symptoms, the association of somatic and anxiety symptoms (both concurrently and prospectively)

Human pluripotent stem cells, including cloned embryonic and induced pluripotent stem cells, offer a limitless cellular source for regenerative medicine. However, their derivation efficiency is limited, and a large proportion of cells are arrested during reprogramming. In the current study, we explored chromosome microdeletion/duplication in arrested and established reprogrammed cells. Our results show that aneuploidy induced by somatic cell nuclear transfer technology is a key factor in the developmental failure of cloned human embryos and primary colonies from implanted cloned blastocysts and that expression patterns of apoptosis-related genes are dynamically altered. Overall, ~20%53% of arrested primary colonies in induced plurpotent stem cells displayed aneuploidy, and upregulation of P53 and Bax occurred in all arrested primary colonies. Interestingly, when somatic cells with pre-existing chromosomal mutations were used as donor cells, no cloned blastocysts were obtained, and additional chromosomal mutations were detected in the resulting iPS cells following long-term culture, which was not observed in the two iPS cell lines with normal karyotypes. In conclusion, aneuploidy induced by the reprogramming process restricts the derivation of pluripotent stem cells, and, more importantly, pre-existing chromosomal mutations enhance the risk of genome instability, which limits the clinical utility of these cells. PMID:25965553

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis. PMID:21897352

Laser scanning microscopy provides high-resolution nondestructive in vivo imaging to capture specific structures that have been fluorescently labeled, such as cellular nuclei and membranes, throughout early zebrafish embryogenesis. An increasingly challenging problem biologists must face is how to effectively explore, follow, and study the thousands of cells contained in the resulting time-varying volume data that are large in space, time, and variable domain. Visual data explorations, such as direct volume rendering, have been successfully used for the analysis of volumetric data. However, visualizing large-scale time-varying fields remains a challenging problem. In this paper we present a novel Focus+Context animated volume rendering. The technique is based on the distance map of objects of interest and on a scene graph architecture. We demonstrate that distance map driven volume rendering, implemented in modern graphics hardware, is suited to generate run time and interactive representations such as ghosted rendering and cut-away rendering. The experimental results on zebrafish embryogenesis data demonstrate that the technique is suited to uncover and to analyze biological events, such as organogenesis, contained in time-varying volumetric dataset. PMID:20171844

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection. PMID:26131626

We report, for the first time, a non-syndromic infant with a reversible myeloproliferative disease that harbors a germline hereditary thrombopoietin (THPO) gene mutation, a condition that is known to induce familial thrombocytosis at increasing age. In order to investigate whether somatic THPO gene mutations play a role in sporadic pediatric myeloproliferative diseases, we performed a mutation screening of a large representative cohort of pediatric acute myeloid leukemia, myeloid leukemia of Down syndrome, and juvenile myelomonocytic leukemia samples and show that gain-of-function THPO mutations are extremely rare in sporadic pediatric myeloproliferative diseases. PMID:25728710

Cancer is a complex disease of the genome exhibiting myriad somatic mutations, from single nucleotide changes to various chromosomal rearrangements. The technological advances of next-generation sequencing enable high-throughput ...

The study is concerned the design of new assays that may detect rare somatic mutations in nuclear and mitochondrial DNA, which may increase upon exposure to mutagens, and thus become a marker of human exposure to such mutagens. Two assays for somatic mutation were presented, one for mitochondrial DNA deletions which was developed by the author, and one for deletions of the ADA gene which resides in the nucleus.

all groups on all measures TABLE 3: Means of measures by group 29 30 INTRODUCTION The Role of Catastrophizing in Somatic Illness That stress has a role in the etiology and mai. ntenance of pain and somatic distress is widely accepted. Interest... the experience of pain and to reduce perception of pain or distress (Turk 8 Rudy, 1986). Catastrophizing may be a component of a general coping style or a narrowly focused strategy used only with specific stressors or in certain situations (Wickramasekera...

Transposons have provided important genetic tools for functional genomic screens in lower eukaryotes but have proven less useful in higher eukaryotes because of their low transposition frequency. Here we show that Sleeping Beauty (SB), a member of the Tc1\\/mariner class of transposons, can be mobilized in mouse somatic cells at frequencies high enough to induce embryonic death and cancer in

Uniparental transcripts during embryogenesis may arise due to gamete delivery during fertilization or genomic imprinting. Such transcripts have been found in a number of plant species and appear critical for the early development of embryo or endosperm in seeds. Although the regulatory expression mechanism and function of these genes in embryogenesis require further elucidation, recent studies suggest stage-specific and highly dynamic features that might be essential for critical developmental events such as zygotic division and cell fate determination during embryogenesis. Here, we summarize the current work in this field and discuss future research directions. PMID:25566300

Plant growth regulator (PGR) crosstalk and interaction with the plants genotype and environmental factors play a crucial role in microspore embryogenesis (ME), controlling microspore-derived embryo differentiation and development as well as haploid/doubled haploid plant regeneration. The complexity of the PGR network which could exist at the level of biosynthesis, distribution, gene expression or signaling pathways, renders the creation of an integrated model of ME-control crosstalk impossible at present. However, the analysis of the published data together with the results received recently with the use of modern analytical techniques brings new insights into hormonal regulation of this process. This review presents a short historical overview of the most important milestones in the recognition of hormonal requirements for effective ME in the most important crop plant species and complements it with new concepts that evolved over the last decade of ME studies. PMID:26113852

The distribution of some grapevine viruses in flower explants, embryogenic and non-embryogenic calli, single somatic embryos\\u000a and plants regenerated from embryogenic cultures was investigated by RT-PCR and ELISA. Immature anthers and ovaries of the\\u000a cultivars Grignolino infected by GRSPaV, GLRaV-1 and GVA, Müller-Thurgau infected by GRSPaV and GLRaV-3 and Bosco infected\\u000a by GRSPaV were cultivated on media inducing indirect somatic

Nucleosome organization at promoter regions plays an important role in regulating gene activity. Genome-wide studies in yeast, flies, worms, mammalian embryonic stem cells and transformed cell lines have found well-positioned nucleosomes flanking a nucleosome depleted region (NDR) at transcription start sites. This nucleosome arrangement depends on DNA sequence (cis-elements) as well as DNA binding factors and ATP-dependent chromatin modifiers (trans-factors). However, little is understood about how the nascent embryonic genome positions nucleosomes during development. This is particularly intriguing since the embryonic genome must undergo a broad reprogramming event upon fusion of sperm and oocyte. Using four stages of early embryonic zebrafish development, we map nucleosome positions at the promoter region of 37 zebrafish hox genes. We find that nucleosome arrangement at the hox promoters is a progressive process that takes place over several stages. At stages immediately after fertilization, nucleosomes appear to be largely disordered at hox promoter regions. At stages after activation of the embryonic genome, nucleosomes are detectable at hox promoters, with positions becoming more uniform and more highly occupied. Since the genomic sequence is invariant during embryogenesis, this progressive change in nucleosome arrangement suggests that trans-factors play an important role in organizing nucleosomes during embryogenesis. Separating hox genes into expressed and non-expressed groups shows that expressed promoters have better positioned and occupied nucleosomes, as well as distinct NDRs, than non-expressed promoters. Finally, by blocking the retinoic acid-signaling pathway, we disrupt early hox gene transcription, but observe no effect on nucleosome positions, suggesting that active hox transcription is not a driving force behind the arrangement of nucleosomes at the promoters of hox genes during early development. PMID:23671670

BACKGROUND Although there is increasing recognition of the role of somatic mutations in genetic disorders, the prevalence of somatic mutations in neurodevelopmental disease and the optimal techniques to detect somatic mosaicism have not been systematically evaluated. METHODS Using a customized panel of known and candidate genes associated with brain malformations, we applied targeted high-coverage sequencing (depth, ?200×) to leukocyte-derived DNA samples from 158 persons with brain malformations, including the double-cortex syndrome (subcortical band heterotopia, 30 persons), polymicrogyria with megalencephaly (20), periventricular nodular heterotopia (61), and pachygyria (47). We validated candidate mutations with the use of Sanger sequencing and, for variants present at unequal read depths, subcloning followed by colony sequencing. RESULTS Validated, causal mutations were found in 27 persons (17%; range, 10 to 30% for each phenotype). Mutations were somatic in 8 of the 27 (30%), predominantly in persons with the double-cortex syndrome (in whom we found mutations in DCX and LIS1), persons with periventricular nodular heterotopia (FLNA), and persons with pachygyria (TUBB2B). Of the somatic mutations we detected, 5 (63%) were undetectable with the use of traditional Sanger sequencing but were validated through subcloning and subsequent sequencing of the subcloned DNA. We found potentially causal mutations in the candidate genes DYNC1H1, KIF5C, and other kinesin genes in persons with pachygyria. CONCLUSIONS Targeted sequencing was found to be useful for detecting somatic mutations in patients with brain malformations. High-coverage sequencing panels provide an important complement to whole-exome and whole-genome sequencing in the evaluation of somatic mutations in neuropsychiatric disease. (Funded by the National Institute of Neurological Disorders and Stroke and others.) PMID:25140959

Early stages of vertebrate embryogenesis are characterized by a remarkable series of shape changes. The resulting morphological complexity is driven by molecular, cellular, and tissue-scale biophysical alterations. Operating at the cellular level...

governed by fibroblast growth factor during embryogenesis. Nat. Neurosci. 2, 246­253 11 Raballo, R. In a modern society, we are constantly exposed to a noisy environment: for example, while driving a car during

Summary Reprogramming of somatic cells produces induced pluripotent stem cells (iPSCs) that are invaluable resources for biomedical research. Here, we extended the previous transcriptome studies by performing RNA-seq on cells defined by a combination of multiple cellular surface markers. We found that transcriptome changes during early reprogramming occur independently from the opening of closed chromatin by OCT4, SOX2, KLF4, and MYC (OSKM). Furthermore, our data identify multiple spliced forms of genes uniquely expressed at each progressive stage of reprogramming. In particular, we found a pluripotency-specific spliced form of CCNE1 that is specific to human and significantly enhances reprogramming. In addition, single nucleotide polymorphism (SNP) expression analysis reveals that monoallelic gene expression is induced in the intermediate stages of reprogramming, while biallelic expression is recovered upon completion of reprogramming. Our transcriptome data provide unique opportunities in understanding human iPSC reprogramming. PMID:26004630

Reprogramming of somatic cells produces induced pluripotent stem cells (iPSCs) that are invaluable resources for biomedical research. Here, we extended the previous transcriptome studies by performing RNA-seq on cells defined by a combination of multiple cellular surface markers. We found that transcriptome changes during early reprogramming occur independently from the opening of closed chromatin by OCT4, SOX2, KLF4, and MYC (OSKM). Furthermore, our data identify multiple spliced forms of genes uniquely expressed at each progressive stage of reprogramming. In particular, we found a pluripotency-specific spliced form of CCNE1 that is specific to human and significantly enhances reprogramming. In addition, single nucleotide polymorphism (SNP) expression analysis reveals that monoallelic gene expression is induced in the intermediate stages of reprogramming, while biallelic expression is recovered upon completion of reprogramming. Our transcriptome data provide unique opportunities in understanding human iPSC reprogramming. PMID:26004630

A broad spectrum of genetic and epigenetic changes is induced by wide hybridization and subsequent polyploidization, but the timing of these events remains obscure because early hybrid cells are very difficult to harvest and analyze. Here, we used both cytological and genetic marker approaches to analyze the constitution of very young somatic hybrid cells between japonica rice (Oryza sativa L. subsp japonica) and indica rice (Oryza sativa L. subsp indica) and between japonica rice and bread wheat (Triticum aestivum L.). Chromatin elimination, simple sequence repeats, and retrotransposon profile deletions were already apparent within six days of the fusion event. The evidence we have presented suggests that genomic changes induced by genomic shock occur soon after the formation of hybrid cells. PMID:24668681

Background Creatine (Cr) is synthesized by a two-step mechanism involving arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), and is taken up by cells through a specific Cr transporter, CT1. Recently, genetic defects of this pathway have been described, that lead to Cr deficiency, neurological symptoms in early infancy and severe neurodevelopmental delay. To investigate the involvement of Cr synthesis and uptake pathways during embryonic development, we determined the spatiotemporal expression of AGAT, GAMT and CT1 during the rat embryogenesis, at the mRNA and protein level. Results We show that AGAT and GAMT are expressed in hepatic primordium as soon as 12.5 days, then progressively acquire their adult pattern of expression, with high levels of AGAT in kidney and pancreas, and high levels of GAMT in liver and pancreas. AGAT and CT1 are prominent in CNS, skeletal muscles and intestine, where they appear earlier than GAMT. High levels of CT1 are found in epithelia. Conclusion Our results suggest that de novo synthesis of Cr by AGAT and GAMT, as well as cellular Cr uptake by CT1, are essential during embryonic development. This work provides new clues on how creatine can be provided to developing tissues, and suggests that Cr deficiencies might induce irreversible damages already in utero, particularly on the nervous system. PMID:15918910

Background Lymphatic filariasis and onchocerciasis are two chronic diseases mediated by parasitic filarial worms causing long term disability and massive socioeconomic problems. Filariae are transmitted by blood-feeding mosquitoes that take up the first stage larvae from an infected host and deliver it after maturation into infective stage to a new host. After closure of vector control programs, disease control relies mainly on mass drug administration with drugs that are primarily effective against first stage larvae and require many years of annual/biannual administration. Therefore, there is an urgent need for alternative treatment ways, i.e. other effective drugs or vaccines. Methodology/Principal Findings Using the Litomosoides sigmodontis murine model of filariasis we demonstrate that immunization with microfilariae together with the adjuvant alum prevents mice from developing high microfilaraemia after challenge infection. Immunization achieved 70% to 100% protection in the peripheral blood and in the pleural space and furthermore strongly reduced the microfilarial load in mice that remained microfilaraemic. Protection was associated with the impairment of intrauterine filarial embryogenesis and with local and systemic microfilarial-specific host IgG, as well as IFN-? secretion by host cells from the site of infection. Furthermore immunization significantly reduced adult worm burden. Conclusions/Significance Our results present a tool to understand the immunological basis of vaccine induced protection in order to develop a microfilariae-based vaccine that reduces adult worm burden and prevents microfilaraemia, a powerful weapon to stop transmission of filariasis. PMID:22413031

Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (approximately 3-4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink. PMID:16584722

DNA damage in somatic cells originates from both environmental and endogenous sources, giving rise to mutations through multiple mechanisms. When these mutations affect the function of critical genes, cancer may ensue. Although identifying genomic subsets of mutated genes may inform therapeutic options, a systematic survey of tumor mutational spectra is required to improve our understanding of the underlying mechanisms of mutagenesis involved in cancer etiology. Recent studies have presented genome-wide sets of somatic mutations as a 96-element vector, a procedure that only captures the immediate neighbors of the mutated nucleotide. Herein, we present a 32 × 12 mutation matrix that captures the nucleotide pattern two nucleotides upstream and downstream of the mutation. A somatic autosomal mutation matrix (SAMM) was constructed from tumor-specific mutations derived from each of 909 individual cancer genomes harboring a total of 10,681,843 single-base substitutions. In addition, mechanistic template mutation matrices (MTMMs) representing oxidative DNA damage, ultraviolet-induced DNA damage, (5m)CpG deamination, and APOBEC-mediated cytosine mutation, are presented. MTMMs were mapped to the individual tumor SAMMs to determine the maximum contribution of each mutational mechanism to the overall mutation pattern. A Manhattan distance across all SAMM elements between any two tumor genomes was used to determine their relative distance. Employing this metric, 89.5 % of all tumor genomes were found to have a nearest neighbor from the same tissue of origin. When a distance-dependent 6-nearest neighbor classifier was used, 86.9 % of all SAMMs were assigned to the correct tissue of origin. Thus, although tumors from different tissues may have similar mutation patterns, their SAMMs often display signatures that are characteristic of specific tissues. PMID:26001532

Vascular endothelial cells in the central nervous system(CNS) forma barrier that restricts the movement of molecules and ions between the blood and the brain. This bloodbrain barrier (BBB) is crucial to ensure proper neuronal function and protect the CNS from injury and disease1. Transplantation studies have demonstrated that the BBB is not intrinsic to the endothelial cells, but is induced by interactions with the neural cells2. Owing to the close spatial relationship between astrocytes and endothelial cells, it has been hypothesized that astrocytes induce this critical barrier postnatally3, but the timing of BBB formation has been controversial49. Here we demonstrate that the barrier is formed during embryogenesis as endothelial cells invade the CNS and pericytes are recruited to the nascent vessels, over a week before astrocyte generation. Analysing mice with null and hypomorphic alleles of Pdgfrb, which have defects in pericyte generation, we demonstrate that pericytes are necessary for the formation of the BBB, and that absolute pericyte coverage determines relative vascular permeability. We demonstrate that pericytes regulate functional aspects of the BBB, including the formation of tight junctions and vesicle trafficking in CNS endothelial cells. Pericytes do not induce BBB-specific gene expression in CNS endothelial cells, but inhibit the expression of molecules that increase vascular permeability and CNS immune cell infiltration. These data indicate that pericyteendothelial cell interactions are critical to regulate the BBB during development, and disruption of these interactions may lead to BBB dysfunction and neuroinflammation during CNS injury and disease. PMID:20944625

Background Many aspects of autoimmune disease are not well understood, including the specificities of autoimmune targets, and patterns of co-morbidity and cross-heritability across diseases. Prior work has provided evidence that somatic mutation caused by gene conversion and deletion at segmentally duplicated loci is relevant to several diseases. Simple tandem repeat (STR) sequence is highly mutable, both somatically and in the germ-line, and somatic STR mutations are observed under inflammation. Results Protein-coding genes spanning STRs having markers of mutability, including germ-line variability, high total length, repeat count and/or repeat similarity, are evaluated in the context of autoimmunity. For the initiation of autoimmune disease, antigens whose autoantibodies are the first observed in a disease, termed primary autoantigens, are informative. Three primary autoantigens, thyroid peroxidase (TPO), phogrin (PTPRN2) and filaggrin (FLG), include STRs that are among the eleven longest STRs spanned by protein-coding genes. This association of primary autoantigens with long STR sequence is highly significant (). Long STRs occur within twenty genes that are associated with sixteen common autoimmune diseases and atherosclerosis. The repeat within the TTC34 gene is an outlier in terms of length and a link with systemic lupus erythematosus is proposed. Conclusions The results support the hypothesis that many autoimmune diseases are triggered by immune responses to proteins whose DNA sequence mutates somatically in a coherent, consistent fashion. Other autoimmune diseases may be caused by coherent somatic mutations in immune cells. The coherent somatic mutation hypothesis has the potential to be a comprehensive explanation for the initiation of many autoimmune diseases. PMID:24988487

Nuclear reprogramming of somatic cells with ectopic stemness factors to bioengineer pluripotent autologous stem cells signals a new era in regenerative medicine. The study of developmental biology has provided a roadmap for cardiac differentiation from embryonic tissue formation to adult heart muscle rejuvenation. Understanding the molecular mechanisms of stem-cell-derived cardiogenesis enables the reproducible generation, isolation, and monitoring of progenitors that have the capacity to recapitulate embryogenesis and differentiate into mature cardiac tissue. With the advent of induced pluripotent stem (iPS) cell technology, patient-specific stem cells provide a reference point to systematically decipher cardiogenic differentiation through discrete stages of development. Interrogation of iPS cells and their progeny from selected cohorts of patients is an innovative approach towards uncovering the molecular mechanisms of disease. Thus, the principles of cardiogenesis can now be applied to regenerative medicine in order to optimize personalized therapeutics, diagnostics, and discovery-based science for the development of novel clinical applications. PMID:20956984

Somatic cell counts (log, base 2)and rates of infection in first and subsequent lactations were examined by classes of somatic cell count in initial test day of first lactation to determine if cows with initially low\\

Pyramidal cells of the apteronotid ELL have been shown to display a characteristic mechanism of burst discharge, which has been shown to play an important role in sensory coding. This form of bursting depends on a reciprocal dendro-somatic interaction, in which discharge of a somatic spike causes a dendritic spike, which in turn contributes a dendro-somatic current flow to create

Embryologists working with livestock species were the pioneers in the field of reprogramming by somatic cell nuclear transfer (SCNT). Without the "Dolly experiment," the field of cellular reprogramming would have been slow and induced plutipotent cells (iPSCs) would not have been conceived. The major drive of the work in mammalian cloning was the interest of the breeding industry to propagate superior genotypes. Soon it was realized that the properties of oocytes could be used also to clone endangered mammalian species or to reprogram the genomes of unrelated species through what is known as interspecies (i) SCNT, using easily available oocytes of livestock species. iSCNT for cloning animals works only for species that can interbreed, and experiments with taxonomically distant species have not been successful in obtaining live births or deriving embryonic stem cell (ESC) lines to be used for regenerative medicine. There are controversial reports in the literature, but in most cases these experiments have underlined some of the cellular and molecular mechanisms that are incomplete during cell nucleus reprogramming, including the failure to organize nucleoli, silence somatic cell genes, activate the embryonic genome, and resume mitochondrial replication and function, thus indicating nucleus-cytoplasmic incompatibility. PMID:24033141

Somatic cell reprogramming has dramatically changed stem cell research in recent years. The high pace of new findings in the field and an ever increasing amount of data from new high throughput techniques make it challenging to isolate core principles of the process. In order to analyze such mechanisms, we developed an abstract mechanistic model of a subset of the known regulatory processes during cell differentiation and production of induced pluripotent stem cells. This probabilistic Boolean network describes the interplay between gene expression, chromatin modifications, and DNA methylation. The model incorporates recent findings in epigenetics and partially reproduces experimentally observed reprogramming efficiencies and changes in methylation and chromatin remodeling. It enables us to investigate, how the temporal progression of the process is regulated. It also explicitly includes the transduction of factors using viral vectors and their silencing in reprogrammed cells, since this is still a standard procedure in somatic cell reprogramming. Based on the model we calculate an epigenetic landscape for probabilities of cell states. Simulation results show good reproduction of experimental observations during reprogramming, despite the simple structure of the model. An extensive analysis and introduced variations hint toward possible optimizations of the process that could push the technique closer to clinical applications. Faster changes in DNA methylation increase the speed of reprogramming at the expense of efficiency, while accelerated chromatin modifications moderately improve efficiency. PMID:22754535

Reprogramming to pluripotency is a low-efficiency process at the population level. Despite notable advances to molecularly characterize key steps, several fundamental aspects remain poorly understood, including when the potential to reprogram is first established. Here, we apply live-cell imaging combined with a novel statistical approach to infer when somatic cells become fated to generate downstream pluripotent progeny. By tracing cell lineages from several divisions before factor induction through to pluripotent colony formation, we find that pre-induction sister cells acquire similar outcomes. Namely, if one daughter cell contributes to a lineage that generates induced pluripotent stem cells (iPSCs), its paired sibling will as well. This result suggests that the potential to reprogram is predetermined within a select subpopulation of cells and heritable, at least over the short term. We also find that expanding cells over several divisions prior to factor induction does not increase the per-lineage likelihood of successful reprogramming, nor is reprogramming fate correlated to neighboring cell identity or cell-specific reprogramming factor levels. By perturbing the epigenetic state of somatic populations with Ezh2 inhibitors prior to factor induction, we successfully modulate the fraction of iPSC-forming lineages. Our results therefore suggest that reprogramming potential may in part reflect preexisting epigenetic heterogeneity that can be tuned to alter the cellular response to factor induction. PMID:25600117

Stem cells are unique pools of cells that are crucial for embryonic development and maintenance of adult tissue homeostasis. The landmark Nobel Prize winning research by Yamanaka and colleagues to induce pluripotency in somatic cells has reshaped the field of stem cell research. The complications related to the usage of pluripotent embryonic stem cells (ESCs) in human medicine, particularly ESC isolation and histoincompatibility were bypassed with induced pluripotent stem cell (iPSC) technology. The human iPSCs can be used for studying embryogenesis, disease modeling, drug testing and regenerative medicine. iPSCs can be diverted to different cell lineages using small molecules and growth factors. In this review we have focused on iPSC differentiation towards cardiac and neuronal lineages. Moreover, we deal with the use of iPSCs in regenerative medicine and modeling diseases like myocardial infarction, Timothy syndrome, dilated cardiomyopathy, Parkinson's, Alzheimer's and Huntington's disease. Despite the promising potential of iPSCs, genome contamination and low efficacy of cell reprogramming remain significant challenges. PMID:25689424

Childhood exposure to trauma has been associated with increased rates of somatic symptoms (SS), which may contribute to diminished daily functioning. One hundred and sixty-one children residing at a residential treatment home who had experienced neglect and/or abuse were administered the Trauma Symptom Checklist for Children (TSCC), the

Peripheral sensory ganglia contain somata of afferent fibres conveying somatosensory inputs to the central nervous system. Growing evidence suggests that the somatic/perisomatic region of sensory neurons can influence peripheral sensory transmission. Control of resting membrane potential (Erest) is an important mechanism regulating excitability, but surprisingly little is known about how Erest is regulated in sensory neuron somata or how changes in somatic/perisomatic Erest affect peripheral sensory transmission. We first evaluated the influence of several major ion channels on Erest in cultured small-diameter, mostly capsaicin-sensitive (presumed nociceptive) dorsal root ganglion (DRG) neurons. The strongest and most prevalent effect on Erest was achieved by modulating M channels, K2P and 4-aminopiridine-sensitive KV channels, while hyperpolarization-activated cyclic nucleotide-gated, voltage-gated Na+, and T-type Ca2+ channels to a lesser extent also contributed to Erest. Second, we investigated how varying somatic/perisomatic membrane potential, by manipulating ion channels of sensory neurons within the DRG, affected peripheral nociceptive transmission in vivo. Acute focal application of M or KATP channel enhancers or a hyperpolarization-activated cyclic nucleotide-gated channel blocker to L5 DRG in vivo significantly alleviated pain induced by hind paw injection of bradykinin. Finally, we show with computational modelling how somatic/perisomatic hyperpolarization, in concert with the low-pass filtering properties of the t-junction within the DRG, can interfere with action potential propagation. Our study deciphers a complement of ion channels that sets the somatic Erest of nociceptive neurons and provides strong evidence for a robust filtering role of the somatic and perisomatic compartments of peripheral nociceptive neuron. PMID:25168672

To gain insights into coordinated lineage-specification and morphogenetic processes during early embryogenesis, here we report a systematic identification of transcriptional programs mediated by a key developmental regulator--Brachyury. High-resolution chromosomal localization mapping of Brachyury by ChIP sequencing and ChIP-exonuclease revealed distinct sequence signatures enriched in Brachyury-bound enhancers. A combination of genome-wide in vitro and in vivo perturbation analysis and cross-species evolutionary comparison unveiled a detailed Brachyury-dependent gene-regulatory network that directly links the function of Brachyury to diverse developmental pathways and cellular housekeeping programs. We also show that Brachyury functions primarily as a transcriptional activator genome-wide and that an unexpected gene-regulatory feedback loop consisting of Brachyury, Foxa2, and Sox17 directs proper stem-cell lineage commitment during streak formation. Target gene and mRNA-sequencing correlation analysis of the T(c) mouse model supports a crucial role of Brachyury in up-regulating multiple key hematopoietic and muscle-fate regulators. Our results thus chart a comprehensive map of the Brachyury-mediated gene-regulatory network and how it influences in vivo developmental homeostasis and coordination. PMID:24616493

Unlike animals, where the germline is established early during embryogenesis, plants set aside their reproductive lineage late in development in dedicated floral organs. The specification of pollen mother cells (PMC) committed to meiosis takes place in the sporogenous tissue in anther locules and marks the somatic-to-reproductive cell fate transition toward the male reproductive lineage. Here we show that Arabidopsis PMC differentiation is accompanied by large-scale changes in chromatin organization. This is characterized by significant increase in nuclear volume, chromatin decondensation, reduction in heterochromatin, eviction of linker histones and the H2AZ histone variant. These structural alterations are accompanied by dramatic, quantitative changes in histone modifications levels compared to that of surrounding somatic cells that do not share a sporogenic fate. All these changes are highly reminiscent of those we have formerly described in female megaspore mother cells (MMC). This indicates that chromatin reprogramming is a common underlying scenario in the somatic-to-reproductive cell fate transition in both male and female lineages. PMID:25972887

Background Late Embryogenesis Abundant Proteins database (LEAPdb) contains resource regarding LEAP from plants and other organisms. Although LEAP are grouped into several families, there is no general consensus on their definition and on their classification. They are associated with abiotic stress tolerance, but their actual function at the molecular level is still enigmatic. The scarcity of 3-D structures for LEAP remains a handicap for their structure-function relationships analysis. Finally, the growing body of published data about LEAP represents a great amount of information that needs to be compiled, organized and classified. Results LEAPdb gathers data about 8 LEAP sub-families defined by the PFAM, the Conserved Domain and the InterPro databases. Among its functionalities, LEAPdb provides a browse interface for retrieving information on the whole database. A search interface using various criteria such as sophisticated text expression, amino acids motifs and other useful parameters allows the retrieving of refined subset of entries. LEAPdb also offers sequence similarity search. Information is displayed in re-ordering tables facilitating the analysis of data. LEAP sequences can be downloaded in three formats. Finally, the user can submit his sequence(s). LEAPdb has been conceived as a user-friendly web-based database with multiple functions to search and describe the different LEAP families. It will likely be helpful for computational analyses of their structure - function relationships. Conclusions LEAPdb contains 769 non-redundant and curated entries, from 196 organisms. All LEAP sequences are full-length. LEAPdb is publicly available at http://forge.info.univ-angers.fr/~gh/Leadb/index.php. PMID:20359361

The bdelloid rotifer Adineta ricciae is an asexual microinvertebrate that can survive desiccation by entering an ametabolic state known as anhydrobiosis. Two late embryogenesis abundant (LEA) proteins, ArLEA1A and ArLEA1B, have been hypothesized to contribute to desiccation tolerance in these organisms, since in vitro assays suggest that ArLEA1A and ArLEA1B stabilize desiccation-sensitive proteins and membranes, respectively. To examine their functions in vivo, it is important to analyse the cellular distribution of the bdelloid LEA proteins. Bioinformatics predicted their translocation into the endoplasmic reticulum (ER) via an N-terminal ER translocation signal and persistence in the same compartment via a variant C-terminal retention signal sequence ATEL. We assessed the localization of LEA proteins in bdelloids and in a mammalian cell model. The function of the N-terminal sequence of ArLEA1A and ArLEA1B in mediating ER translocation was verified, but our data showed that, unlike classical ER-retention signals, ATEL allows progression from the ER to the Golgi and limited secretion of the proteins into the extracellular medium. These results suggest that the N-terminal ER translocation signal and C-terminal ATEL sequence act together to regulate the distribution of rotifer LEA proteins within intracellular vesicular compartments, as well as the extracellular space. We speculate that this mechanism allows a small number of LEA proteins to offer protection to a large number of desiccation-sensitive molecules and structures both inside and outside cells in the bdelloid rotifer. PMID:22837450

Focal cortical dysplasia type II (FCDII) is a sporadic developmental malformation of the cerebral cortex characterized by dysmorphic neurons, dyslamination and medically refractory epilepsy. It has been hypothesized that FCD is caused by somatic mutations in affected regions. Here, we used deep whole-exome sequencing (read depth, 412-668×) validated by site-specific amplicon sequencing (100-347,499×) in paired brain-blood DNA from four subjects with FCDII and uncovered a de novo brain somatic mutation, mechanistic target of rapamycin (MTOR) c.7280T>C (p.Leu2427Pro) in two subjects. Deep sequencing of the MTOR gene in an additional 73 subjects with FCDII using hybrid capture and PCR amplicon sequencing identified eight different somatic missense mutations found in multiple brain tissue samples of ten subjects. The identified mutations accounted for 15.6% of all subjects with FCDII studied (12 of 77). The identified mutations induced the hyperactivation of mTOR kinase. Focal cortical expression of mutant MTOR by in utero electroporation in mice was sufficient to disrupt neuronal migration and cause spontaneous seizures and cytomegalic neurons. Inhibition of mTOR with rapamycin suppressed cytomegalic neurons and epileptic seizures. This study provides, to our knowledge, the first evidence that brain somatic activating mutations in MTOR cause FCD and identifies mTOR as a treatment target for intractable epilepsy in FCD. PMID:25799227

Maturation of black spruce somatic embryos in sealed and vented microenvironments was investigated. The sealed microenvironment\\u000a induced a larger number of well-formed mature embryos and less precocious germination than the vented microenvironment. Maturation\\u000a rate of somatic embryos was not changed either by injection of ethylene into the culture vessel or by its removal by potassium\\u000a permanganate traps. Increased as well

The advance in technological tools for massively parallel, high-throughput sequencing of DNA has enabled the comprehensive characterization of somatic mutations in large number of tumor samples. Here, we review recent cancer genomic studies that have assembled emerging views of the landscapes of somatic mutations through deep sequencing analyses of the coding exomes and whole genomes in various cancer types. We discuss the comparative genomics of different cancers, including mutation rates, spectrums, and roles of environmental insults that influence these processes. We highlight the developing statistical approaches used to identify significantly mutated genes, and discuss the emerging biological and clinical insights from such analyses as well as the challenges ahead translating these genomic data into clinical impacts. PMID:24022702

Growth is a polygenic trait that is under the influence of multiple physiological pathways regulating energy metabolism and muscle growth. Among the possible growth-regulating pathways in vertebrates, components of the somatotropic axis are thought to have the greatest influence. There is growing body of literature focusing on the somatotropic axis and its role regulating growth in fish. This includes research into growth hormone, upstream hypothalamic hormones, insulin-like growth factors, and downstream signaling molecules. Many of these signals have both somatic effects stimulating the growth of tissues and metabolic effects that play a role in nutrient metabolism. Signals of other endocrine axes exhibit profound effects on the function of the somatotropic axis in vivo. In this review we highlight recent advances in our understanding of the teleost fish endocrine somatotropic axis, including emerging research using genetic modified models. These studies have revealed new aspects and challenges associated with regulation of the important steps of somatic growth. PMID:25655896

Reprogramming somatic cells to become induced pluripotent stem cells (iPSCs) by using defined factors represents an important breakthrough in biology and medicine, yet remains inefficient and poorly understood. We therefore devised synthetic factors by fusing the VP16 transactivation domain to OCT4 (also known as Pou5f1), NANOG and SOX2, respectively. These synthetic factors could reprogramme both mouse and human fibroblasts with

Somatic hybrids were produced by PEG-induced symmetric and asymmetric protoplast fusions in order to transfer resistance to\\u000a Alternaria brassicicola, A. brassicae, Phoma\\u000a lingam, Plasmodiophora brassicae and Turnip mosaic virus (TuMV) into Brassica oleracea var. capitata (cv. Toskama) and botrytis (cv. Korso). As resistance donors, ten species belonging to several genera of the family Brassicaceae including wild relatives\\u000a were used. Of

The induction of cytogenetic effects by inhalation of ethylene oxide was tested in bone marrow cells and primary spermatocytes at diakinesis-metaphase I cells from mouse after a single treatment (6 h\\/1 day) at 0, 200, 400 and 600 ppm, and multiple treatment (6 h\\/5 days\\/2 weeks) at 0, 200 and 400 ppm. Ethylene oxide induced chromosomal aberrations in both somatic

Human induced pluripotent stem cells (iPSCs) have become an intriguing approach for neurological disease modeling, because neural lineage-specific cell types that retain the donors complex genetics can be established in vitro. The statistical power of these iPSC-based models, however, is dependent on accurate diagnoses of the somatic cell donors; unfortunately, many neurodegenerative diseases are commonly misdiagnosed in live human subjects.

Induced pluripotent stem cells (iPSCs) are generated by directly reprogramming somatic cells by forcing them to express the exogenous transcription factors, Oct4, Sox2, Klf4 and c-Myc (OSKM). These cells could potentially be used in clinical applications and basic research. Here, we explored the molecular role of Sox2 by generating iPSCs that expressed Sox2 at various levels. Low Sox2 (LS) expression

An adult mammal is composed of more than 200 different types of specialized somatic cells whose differentiated state remains stable over the life of the organism. For a long time it was believed that the differentiation process is irreversible, and the transition between the two types of specialized cells is impossible. The possibility of direct conversion of one differentiated cell type to another was first shown in the 80s of the last century in experiments on the conversion of fibroblasts into myoblasts by ectopic expression of the transcription factor MyoD. Surprisingly, this technology has remained unclaimed in cell biology for a long time. Interest in it revived after 200 thanks to the research of Novel Prize winner Shinya Yamanaka who has shown that a small set of transcription factors (Oct4, Sox2, Klf4 and c-Myc) is capable of restoring pluripotency in somatic cells which they lost in the process of differentiation. In 2010, using a similar strategy and the tissue-specific transcription factors Vierbuchen and coauthors showed the possibility of direct conversion of fibroblasts into neurons, i. e. the possibility of transdifferentiation of one type of somatic cells in the other. The works of these authoras were a breakthrough in the field of cell biology and gave a powerful impulse to the development of cell technologies for the needs of regenerative medicine. The present review discusses the main historical discoveries that preceded this work, evaluates the status of the problem and the progress in the development of methods for reprogramming at the moment, describes the main approaches to solving the problems of reprogramming of somatic cells into neuronal, and briefly discusses the prospect of application of reprogramming and transdifferentiation of cells for such important application areas as regenerative medicine, cell replacement therapy and drug screening. PMID:25929128

Although regeneration is widespread among metazoa, the molecular mechanisms have been studied in only a handful of taxa. Of these taxa, fewer still are amenable to studies of embryogenesis. Our understanding of the evolution of regeneration, and its relation to embryogenesis, therefore remains limited. Using ?-catenin as a marker, we investigated the role of canonical Wnt signaling during both regeneration and embryogenesis in the cnidarian Nematostella vectensis. The canonical Wnt signaling pathway is known to play a conserved role in primary axis patterning in triploblasts. Induction of Wnt signaling with alsterpaullone results in ectopic oral tissue during both regeneration and embryogenesis by specifically upregulating ?-catenin expression, as measured by qRTPCR. Our data indicate that canonical Wnt signaling is sufficient for oral patterning during Nematostella regeneration and embryogenesis. These data also contribute to a growing body of literature indicating a conserved role for patterning mechanisms across various developmental modes of metazoans. PMID:22052821

Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since Dolly, the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

In 1997, a cloned sheep "Dolly" was produced by nuclear transfer of somatic cell. The first birth of cloned mice derived from some somatic cells were succeeded in 1998. At present, it is shown that somatic cells, cumulus cells, fibroblasts and Sertoli cells can be used to the study of cloned animal as nuclear donor. In this study investigation was designed to compare with efficiency on the production of cloned embryos by using the microinjection and the electrofusion methods for nuclear transfer. Oocyte enucleation was performed with a micromanipulator. The oocyte was held by holding pipette, and was enucleated using a beveled pipette. Microinjection method: Cell's nucleus injection was carried out by piezo-micromanipulator. Cytochalasin B treated cumulus cell was aspirated into a injection pipette, and was broken its plasma membrane using the injection pipette. Then, the cumulus cell was injected into the enucleated ooplasm directly. Electrofusion method: The cell was aspirated into a beveled pipette, and then an aspirated cell was inserted into perivitelline space. Then, the pair of enucleated oocyte and cell was fused using electrical cell fusion apparatus. The reconstituted embryos were activated after nuclear transfer using St2+. Reconstituted embryos had been produced by the microinjection showed the embryonic development to over 8-cell stages. But, the rate of fragmentation of reconstituted embryos by the microinjection showed a little high rate in comparison with the electrofusion. When some reconstituted embryos by the microinjection were transplanted to pseudopregnant females' oviduct, 9 fetuses were observed at 14 days post coitum. PMID:11329940

Broad phenotypic variations were induced in derivatives of an asymmetric somatic hybridization of bread wheat (Triticum aestivum) and tall wheatgrass (Thinopyrum ponticum Podp); however, how these variations occurred was unknown. We explored the nature of these variations by cytogenetic assays and DNA profiling techniques to characterize six genetically stable somatic introgression lines. Karyotyping results show the six lines similar to their wheat parent, but GISH analysis identified the presence of a number of short introgressed tall wheatgrass chromatin segments. DNA profiling revealed many genetic and epigenetic differences, including sequences deletions, altered regulation of gene expression, changed patterns of cytosine methylation, and the reactivation of retrotransposons. Phenotypic variations appear to result from altered repetitive sequences combined with the epigenetic regulation of gene expression and/or retrotransposon transposition. The extent of genetic and epigenetic variation due to the maintenance of parent wheat cells in tissue culture was assessed and shown to be considerably lower than had been induced in the introgression lines. Asymmetric somatic hybridization provides appropriate material to explore the nature of the genetic and epigenetic variations induced by genomic shock. PMID:25670745

In early plant embryogenesis, the determination of cell fate in the protodermal cell layer is considered to be the earliest event in radial pattern formation. To elucidate the mechanisms of epidermal cell fate determination and radial pattern formation in early rice embryogenesis, we have isolated a GL2-type homeobox gene Roc1 (Rice outermost cell-specific gene1), which is specifically expressed in the protoderm (epidermis). In early rice embryogenesis, cell division occurs randomly and the morphologically distinct layer structure of the protoderm cannot be observed until the embryo reaches more than 100 microm in length. Nonetheless, in situ hybridization analyses revealed that specific expression of Roc1 in the outermost cells is established shortly after fertilization, much earlier than protoderm differentiation. In the regeneration process from callus, the Roc1 gene is also expressed in the outermost cells of callus in advance of tissue and organ differentiation, and occurs independently of whether the cells will differentiate into epidermis in the future or not. Furthermore, this cell-specific Roc1 expression could be induced flexibly in the newly produced outermost cells when we cut the callus. These findings suggest that the expression of Roc1 in the outermost cells may be dependent on the positional information of cells in the embryo or callus prior to the cell fate determination of the protoderm (epidermis). Furthermore, the Roc1 expression is downregulated in the inner cells of ligule, which have previously been determined as protodermal cells, also suggesting that the Roc1 expression is position dependent and that this position dependent Roc1 expression is important also in post-embryonic protoderm (epidermis) differentiation. PMID:11846882

Background Unexplained somatic symptoms are common among trauma survivors. The relationship between trauma and somatization appears to be mediated by posttraumatic stress disorder (PTSD). However, only few studies have focused on what other psychological risk factors may predispose a trauma victim towards developing somatoform symptoms. Methods The present paper examines the predictive value of PTSD severity, dissociation, negative affectivity, depression, anxiety, and feeling incompetent on somatization in a Danish sample of 169 adult men and women who were affected by a series of explosions in a firework factory settled in a residential area. Results Negative affectivity and feelings of incompetence significantly predicted somatization, explaining 42% of the variance. PTSD was significant until negative affectivity was controlled for. Conclusion Negative affectivity and feelings of incompetence significantly predicted somatization in the trauma sample whereas dissociation, depression, and anxiety were not associated with degree of somatization. PTSD as a risk factor was mediated by negative affectivity. PMID:19126224

This paper describes four assays that detect somatic gene mutations in humans: the hypoxanthine-guanine phosphoribosyl transferase assay, the glycophorin A assay, the HLA-A assay, and the sickle cell hemoglobin assay. Somatic gene mutations can be considered a biomarker of carcinogenesis, and assays for somatic mutation may assist epidemiologists in studies that attempt to identify factors associated with increased risks of cancer. Practical aspects of the use of these assays are discussed.

Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

The Wnt cell-cell signaling pathway plays a critical and evolutionarily conserved role in directing cell fates during embryogenesis. In addition, inappropriate activation of the Wnt signal transduction pathway plays a role in a variety of human cancers. Many recent studies of Wnt signaling have provided mechanistic insight into these dual roles. Here we focus on two areas of rapid advance:

BACKGROUND: LEA (late embryogenesis abundant) proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely

Embryos of the inland silverside, Menidia beryllina, were exposed to nominal concentration of 50 ug terbufos 1-1 during the first five days of embryogenesis. ilversides were maintained in clean dilute seawater until 37 days after hatching. adiographs revealed compressed and use v...

Progress is reported on studies on the biochemistry of cotton seed embryogenesis and germination. Data are included on the processing of the stored mRNA of cotton cotyledons during early germination, the characterization of the cotton genome, and the development of the cotton chloroplast protein synthesizing system during germination. (CH)

Neuro-Evolution Using Recombinational Algorithms and Embryogenesis for Robotic Control Thesis individuals helped in the creation of the research presented here, and it would be near impossible to list has been built. #12;v Abstract Control tasks involving dramatic nonlinearities, such as decision

Identification in Pea Seed Mitochondria of a Late-Embryogenesis Abundant Protein Able to Protect) seed mitochondria. PsLEAm revealed typical LEA features such as high hydrophilicity and repeated motifs. The overall results constitute, to our knowledge, the first characterization of a LEA protein in mitochondria

Direct somatic embryo formation (without intervening callus) from garlic clove basal tissue was induced in which the influence of plant growth regulators (PGRs) on various explants was examined. Medium added with 2.0 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) were the most effective PGR combination for somatic embryo induction. It induced embryos directly in 85.5% of the basal clove explant. Callus induction was also obtained from other parts of explant and 2.0 mg/l 2,4-D induced callusing in 86.5% of the inoculated explants. Protein, amino acid and alliin content were measured in callus and in embryos. Somatic embryos had more soluble protein and free amino acid compared to callus. HPTLC analysis revealed that alliin was significantly high in somatic embryos compared to undifferentiated callus tissue; the content was even more in older embryos. The present study of Allium indicates that the event of morphogenetic development including in vitro embryogeny can effectively be analysed by monitoring the changes of biochemical profiles. PMID:20015835

Background A key to understanding the evolution of the nervous system on a large phylogenetic scale is the identification of homologous neuronal types. Here, we focus this search on the sensory and motor neurons of bilaterians, exploiting their well-defined molecular signatures in vertebrates. Sensorimotor circuits in vertebrates are of two types: somatic (that sense the environment and respond by shaping bodily motions) and visceral (that sense the interior milieu and respond by regulating vital functions). These circuits differ by a small set of largely dedicated transcriptional determinants: Brn3 is expressed in many somatic sensory neurons, first and second order (among which mechanoreceptors are uniquely marked by the Brn3+/Islet1+/Drgx+ signature), somatic motoneurons uniquely co-express Lhx3/4 and Mnx1, while the vast majority of neurons, sensory and motor, involved in respiration, blood circulation or digestion are molecularly defined by their expression and dependence on the pan-visceral determinant Phox2b. Results We explore the status of the sensorimotor transcriptional code of vertebrates in mollusks, a lophotrochozoa clade that provides a rich repertoire of physiologically identified neurons. In the gastropods Lymnaea stagnalis and Aplysia californica, we show that homologues of Brn3, Drgx, Islet1, Mnx1, Lhx3/4 and Phox2b differentially mark neurons with mechanoreceptive, locomotory and cardiorespiratory functions. Moreover, in the cephalopod Sepia officinalis, we show that Phox2 marks the stellate ganglion (in line with the respiratory  that is, visceral ancestral role of the mantle, its target organ), while the anterior pedal ganglion, which controls the prehensile and locomotory arms, expresses Mnx. Conclusions Despite considerable divergence in overall neural architecture, a molecular underpinning for the functional allocation of neurons to interactions with the environment or to homeostasis was inherited from the urbilaterian ancestor by contemporary protostomes and deuterostomes. PMID:23631531

Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (<24 h) stronger expression of proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24-48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, ?-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48-72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified biological pathways and key node proteins allow for further functional analysis and genetic manipulation for both the honey bee embryos and other eusocial insects. PMID:24895377

SUMMARY Hypoxia inducible factors (HIFs) are transcription factors controlling energy, iron metabolism, erythropoiesis, and development, and, when dysregulated, contribute to tumorigenesis, cancer progression, and invasion. However, HIF? mutations have not previously been identified in any cancer. Here we report two novel somatic gain-of-function HIF2? mutations in two patients, one presenting with a paraganglioma and a second with both paraganglioma and somatostatinoma. Both mutations were shown to confer increased HIF2? activity and protein half-life. While germline mutations of regulators of HIF?, including VHL and EGLN1, have been reported in pheochromocytomas/paragangliomas, this is the first report of a somatic gain-of-function mutation in HIF. PMID:22931260

The protoplast fusion was induced between Astragalus adsurgens Pall and Agrobacterium rhizogenes-transformed cell line of Medicago Sativa L. by use of PEG method. Putative somatic hybrid cells were selected based on the observation that only intergeneric hybrid cells continued to divide on the DPD medium without any phytohormone while the cells and clusters derived from parental cells did not survive further. After being cultured, the somatic hybrid calli were obtained. Although both of the parental calli were not capable of regenerating , one of the hydrid cell lines recovered the differentiation ability and produced small shoots. Characterizations of the hybrid calli were carried out by examination of chromsome number, opine synthetase assay, isoenzyme and RAPD analyses. PMID:15323417

We have developed a simple, objectively scorable test for the mutagenicity of chemical compounds which can be fed Drosophila melanogaster. The test depends upon the somatic reversion of the X chromosome, recessive eye color mutation, white-ivory (wi) to wild type (w+). Reversions are scored as clones of w+ facets in the wi eyes of eclosing adults. To increase the sensitivity, a tandem quadruplication containing four wi mutations was synthesized. Thus, in homozygous females eight wi mutations are potentially revertible. Six mutagenic compounds, all alkylating agents, all gave positive results at several concentrations tested. Molecular analysis demonstrates that the induced reversions, germinal and somatic, are associated with the loss of 2.9-kilobase DNA duplicated in the wi mutation.

The generation of induced pluripotent stem (iPS) cells holds great promise in regenerative medicine. The use of the transcription factors Oct4, Sox2, Klf4 and c-Myc for reprogramming is extensively documented, but comparatively little is known about soluble molecules promoting reprogramming. Here we identify the secreted cue Netrin-1 and its receptor DCC, described for their respective survival/death functions in normal and oncogenic contexts, as reprogramming modulators. In various somatic cells, we found that reprogramming is accompanied by a transient transcriptional repression of Netrin-1 mediated by an Mbd3/Mta1/Chd4-containing NuRD complex. Mechanistically, Netrin-1 imbalance induces apoptosis mediated by the receptor DCC in a p53-independent manner. Correction of the Netrin-1/DCC equilibrium constrains apoptosis and improves reprogramming efficiency. Our work also sheds light on Netrin-1's function in protecting embryonic stem cells from apoptosis mediated by its receptor UNC5b, and shows that the treatment with recombinant Netrin-1 improves the generation of mouse and human iPS cells. PMID:26154507

The discovery of induced pluripotent stem (iPS) cells provides not only new approaches for cell replacement therapy, but also new ways for drug screening. However, the undefined mechanism and relatively low efficiency of reprogramming have limited the application of iPS cells. In an attempt to further optimize the reprogramming condition, we unexpectedly observed that removing c-Myc from the Oct-4, Sox-2, Klf-4, and c-Myc (OSKM) combination greatly enhanced the generation of iPS cells. The iPS cells generated without c-Myc attained salient pluripotent characteristics and were capable of producing full-term mice through tetraploid complementation. We observed that forced expression of c-Myc induced the expression of many genes involved in cell cycle control and a hyperproliferation state of the mouse embryonic fibroblasts during the early stage of reprogramming. This enhanced proliferation of mouse embryonic fibroblasts correlated negatively to the overall reprogramming efficiency. By applying small molecule inhibitors of cell proliferation at the early stage of reprogramming, we were able to improve the efficiency of iPS cell generation mediated by OSKM. Our data demonstrated that the proliferation rate of the somatic cell plays critical roles in reprogramming. Slowing down the proliferation of the original cells might be beneficial to the induction of iPS cells. PMID:23439651

agronomie: plant genetics and breeding Regulation of somatic embryo development in Norway spruce; Somatic embryos can be used for vegetative propagation of genetically superior material. Additionally, that not all embryogenic cell lines contain embryos that can mature. We are using Norway spruce (Picea abies

Purpose: Of all nonauditory sensory systems, only the somatosensory system seems to be related to tinnitus (eg, temporomandibular joint syndrome and whiplash). The purpose of this study is to describe the distinguishing characteristics of tinnitus associated with somatic events and to use these characteristics to develop a neurological model of somatic tinnitus.Materials and Methods: Case series.Results: Some patients with tinnitus,

The present study was undertaken to evaluate two enucleation methods for somatic cell nuclear transfer (SCNT), and to standardize the optimum number of embryos for transfer to each recipient for canines. Oocytes retrieved from outbreed dogs were reconstructed with adult somatic cells from a male Beagle dog. A total of 134 or 267 oocytes were enucleated either by aspiration or

To identify DNA sequences that target the somatic hypermutation process, the immunoglobulin gene promoter located upstream of the variable (V) region was duplicated upstream of the constant (C) region of a ? transgene. Normally, ? genes are somatically mutated only in the VJ region, but not in the C region. In B cell hybridomas from mice with this ? transgene

The reporting of herd summaries of individual cow somatic cell counts differs between the nine Dairy Records Pro- cessing Centers. The objectives of this paper are 1) to review the present re- porting; 2) to compare the methods used to calculate herd somatic cell counts; and 3) to discuss the epidemio- logic implications of these methods. Estimates of central tendency

Measures per lactation of somatic cell count were developed from monthly test- day observations for 3,966 Holstein cows on the official test option of the Quebec Dairy Herd Analysis Service. Cows were daughters of 99 sires that had five or more daughters in two or more herds. Two lactation periods were considered. One was based on all somatic cell counts

Image processing is widely applied in the field of the biology and medicine domain, however, the study of the milk somatic cell image processing technology is a new topic currently. This paper carries on the pre-processing to the milk somatic cell images firstly. Then in the foundation of the selection of the color space and the analysis based on the

Meningiomas arise from the arachnoid layer of the meninges that surround the brain and spine. They account for over one third of all primary central nervous system tumors in adults and confer a significant risk of location-dependent morbidity due to compression or displacement. A significant increase in risk of meningiomas is associated with neurofibromatosis type 2 (NF2) disease through mutation of the NF2 gene. In addition, approximately 5% of individuals with schwannomatosis disease develop meningiomas, through mutation of the SWI/SNF chromatin remodeling complex subunit, SMARCB1. Recently, a second SWI/SNF complex subunit, SMARCE1, was identified as a cause of clear cell meningiomas, indicating a wider role for this complex in meningioma disease. The sonic hedgehog (SHH)-GLI1 signaling pathway gene, SUFU, has also been identified as the cause of hereditary multiple meningiomas in a large Finnish family. The recent identification of somatic mutations in components of the SHH-GLI1 and AKT1-MTOR signaling pathways indicates the potential for cross talk of these pathways in the development of meningiomas. This review describes the known meningioma predisposition genes and their links to the recently identified somatic mutations. PMID:25857641

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform. PMID:24250782

Somatic cell nuclear transfer (SCNT) cloning is the sole reproductive engineering technology that endows the somatic cell genome with totipotency. Since the first report on the birth of a cloned sheep from adult somatic cells in 1997, many technical improvements in SCNT have been made by using different epigenetic approaches, including enhancement of the levels of histone acetylation in the chromatin of the reconstructed embryos. Although it will take a considerable time before we fully understand the nature of genomic programming and totipotency, we may expect that somatic cell cloning technology will soon become broadly applicable to practical purposes, including medicine, pharmaceutical manufacturing and agriculture. Here we review recent progress in somatic cell cloning, with a special emphasis on epigenetic studies using the laboratory mouse as a model. PMID:23166393

In Coffea arabica L., the development of direct sowing of somatic embryos (SE) in planting substrate, with subsequent nursery production of plants, has promoted the industrialization of somaticembryogenesis. However, plant conversion rates are still low and require improvements to enhance the cost-effectiveness of commercial micropropagation. With the aim of improving plant regeneration from SE, we studied the morphological and histological criteria and water characteristics during germination and plant conversion of zygotic embryos (ZE) and SE. At the cotyledonary stage, SE produced in a 1 l RITA(®) temporary immersion bioreactor (area 55.8 cm(2)) were morphologically similar in size (2-3 mm) but abnormal as compared with mature ZE. Protein and starch reserve levels were extremely low throughout germination and conversion to plantlets, while the water status remained steady [water content (WC) from 76 to 87%, ? from -0.37 to -0.47 MPa, pressure potential from 0.69 to 0.24 MPa]. In ZE, spectacular hydration occurred during the first 3 weeks (WC from 37 to 75%; ? from -6.24 to -1.0 MPa). Cotyledons remained undifferentiated for 10 weeks after sowing. Conversely, after only 3 weeks under germination conditions in a RITA(®) bioreactor, spongy and palisade parenchyma and stomata formed in SE cotyledons. The ZE plant conversion was faster than that of SE (14 vs. 22 weeks) and more efficient (rates 96 vs. 55%), with much more substantial hypocotyl and cotyledon development. The use of a new 5 l MATIS(®) bioreactor (area 355 cm(2)), designed especially to favor embryo dispersion and light transmittance to SE, markedly improved the embryo-to-plantlet conversion rate (91%). These results highlight the morphological heterogeneity and lack of protein reserves in SE at the beginning of the germination phase and marked differences in water characteristics. However, they also reveal high phenotypic plasticity, leading to a highly efficient plantlet conversion rate due to better embryo dispersion and light transmittance in more horizontal bioreactors. PMID:23729274

Prince Rupprecht's larch (Larix principis-rupprechtii Mayr) is a native high-value forest tree species in North China whose clonal propagation through somaticembryogenesis (SE) has the potential to rapidly capture the benefits of breeding or genetic engineering programs and to improve raw material uniformity and quality. To date, research has focused on clarifying the molecular mechanism of SE, but proteomic studies are still in the early stages. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) analysis was performed on three developmental stages of SE in L. principis-rupprechtii in an attempt to identify a wide range of proteins that are regulated differentially during this process. Proteins were extracted and analyzed from the pro-embryogenic mass (PEM), globular embryo (GE), and cotyledon embryo (CE) stages of embryo development. We detected 503 proteins in total and identified 96 proteins expressed differentially during different developmental stages. The identified proteins were analyzed further to provide information about their expression patterns and functions during SE. Four clusters of proteins based on shared expression profiles were generated. Functional analysis showed that proteins involved in primary metabolism, phosphorylation, and oxidation reduction were upregulated during somatic embryo development. This work provides novel insights into the process of larch embryo development in vitro and a basis for further study of the biological process and opportunities for practical application of this knowledge. PMID:25781987

The objective of this paper was to investigate the importance of a genotype x environment interaction (G x E) for somatic cell score (SCS) across levels of bulk milk somatic cell count (BMSCC), number of days in milk (DIM), and their interaction. Variance components were estimated with a model including random regressions for each sire on herd test-day BMSCC, DIM,

A gene expression atlas is an essential resource to quantify and understand the multiscale processes of embryogenesis in time and space. The automated reconstruction of a prototypic 4D atlas for vertebrate early embryos, ...